Therapeutic approaches for treating alzheimer&#39;S disease

ABSTRACT

The present invention relates to compositions and methods for the treatment of Alzheimer&#39;s disease and related disorders. More specifically, the present invention relates to novel combinatorial therapies of Alzheimer&#39;s disease and related disorders. In particular, the invention concerns compounds which, alone or in combination(s), can effectively modulate synapse function and/or angiogenesis and/or cell stress response. The invention also relates to methods of producing a drug or a drug combination for treating Alzheimer&#39;s disease and to methods of treating Alzheimer&#39;s disease or a related disorder.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of International ApplicationNo. PCT/EP2010/066510, filed Oct. 29, 2010, the disclosure of which arehereby incorporated by reference in their entirety, including allfigures, tables and amino acid or nucleic acid sequences.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for thetreatment of Alzheimer's disease (AD) and related disorders. Morespecifically, the present invention relates to novel combinatorialtherapies of Alzheimer's disease and related disorders. In particular,the invention concerns compounds which, alone or in combination(s), caneffectively modulate synapse function and/or angiogenesis and/or cellstress response. The invention also relates to methods of selecting adrug or a drug combination for treating Alzheimer's disease and tomethods of treating Alzheimer's disease or a related disorder.

BACKGROUND OF THE INVENTION

AD is the prototypic cortical dementia characterized by memory deficittogether with dysphasia (language disorder in which there is animpairment of speech and of comprehension of speech), dyspraxia(disability to coordinate and perform certain purposeful movements andgestures in the absence of motor or sensory impairments) and agnosia(ability to recognize objects, persons, sounds, shapes, or smells)attributable to involvement of the cortical association areas. Specialsymptoms such as spastic paraparesis (weakness affecting the lowerextremities) can also be involved (1-4).

Incidence of Alzheimer's disease increases dramatically with the age. ADis at present the most common cause of dementia. It is clinicallycharacterized by a global decline of cognitive function that progressesslowly and leaves end-stage patients bound to bed, incontinent anddependent on custodial care. Death occurs, on average, 9 years afterdiagnosis (5).

The incidence rate of AD increases dramatically with age. United Nationpopulation projections estimate that the number of people older than 80years will approach 370 million by the year 2050. Currently, it isestimated that 50% of people older than age 85 years are afflicted withAD. Therefore, more than 100 million people worldwide will suffer fromdementia in 50 years. The vast number of people requiring constant careand other services will severely affect medical, monetary and humanresources (6).

Memory impairment is the early feature of the disease and involvesepisodic memory (memory for day-today events). Semantic memory (memoryfor verbal and visual meaning) is involved later in the disease. Bycontrast, working memory (short-term memory involving structures andprocesses used for temporarily storing and manipulating information) andprocedural memory (unconscious memory that is long-term memory of skillsand procedure) are preserved until late. As the disease progresses, theadditional features of language impairment, visual perceptual andspatial deficits, agnosias and apraxias emerge.

The classic picture of Alzheimer's disease is sufficientlycharacteristic to allow identification in approximately 80% of cases(7). Nevertheless, clinical heterogeneity does occur and not only isthis important for clinical management but provides further implicationof specific medication treatments for functionally different forms (8).

The pathological hallmark of AD includes amyloid plaques containingbeta-amyloid (Abeta), neurofibrillary tangles (NFT) containing Tau andneuronal and synaptic dysfunction and loss (9-11). For the last decade,two major hypotheses on the cause of AD have been proposed: the “amyloidcascade hypothesis”, which states that the neurodegenerative process isa series of events triggered by the abnormal processing of the AmyloidPrecursor Protein (APP) (12), and the “neuronal cytoskeletaldegeneration hypothesis” (13), which proposes that cytoskeletal changesare the triggering events. The most widely accepted theory explaining ADprogression remains the amyloid cascade hypothesis (14-16) and ADresearchers have mainly focused on determining the mechanisms underlyingthe toxicity associated with Abeta proteins. On contrary, Tau proteinhas received much less attention from the pharmaceutical industry thanamyloid, because of both fundamental and practical concerns. Moreover,synaptic density change is the pathological lesion that best correlateswith cognitive impairment than the two others. Studies have revealedthat the amyloid pathology appears to progress in aneurotransmitter-specific manner where the cholinergic terminals appearmost vulnerable, followed by the glutamatergic terminals and finally bythe GABAergic terminals (11).

SUMMARY OF INVENTION

The purpose of the present invention is to provide new therapeuticapproaches for treating AD and related disorders.

The inventors have identified several drugs which, alone or incombination(s), can effectively affect pathways involved in AD andrepresent a new and effective therapies for the treatment of AD andrelated disorders.

The invention therefore provides novel compositions and methods fortreating AD disease and related disorders.

More particularly, the invention relates to a composition comprising acombination of at least two compounds chosen from the group consistingof aminocaproic acid, acamprosate, amlodipine, argatroban, baclofen,cilostazol, cinacalcet, clopidogrel, dyphylline, fenoldopam,leflunomide, mepacrine, methimazole, phenformin, prilocalne, rifabutin,sulfisoxazole, tadalafil, terbinafine, torasemide, cinnarizine,ciclopirox, eplerenone, carbenoxolone, sulodexide, carbamazine,amobarbital, cefotetan, erythrityl tetranitrate, methyclothiazide,risedronate, enprofylline, oxtriphylline, paramethadione, cefinenoxime,aprindine, etomidate, mitiglinide, benidipine, levosimendan andzonisamide, or salts or prodrugs or derivatives or sustained releaseformulations thereof, for use in the treatment of Alzheimer's disease ora related disorder.

A further object of the present invention relates to a compositioncomprising a combination of at least two compounds chosen from the groupconsisting of aminocaproic acid, acamprosate, amlodipine, argatroban,baclofen, cilostazol, cinacalcet, clopidogrel, dyphylline, fenoldopam,leflunomide, mepacrine, methimazole, phenformin, prilocalne, rifabutin,sulfisoxazole, tadalafil, terbinafine, torasemide, cinnarizine,ciclopirox, eplerenone, carbenoxolone, sulodexide, carbamazine,amobarbital, cefotetan, erythrityl tetranitrate, methyclothiazide,risedronate, enprofylline, oxtriphylline, paramethadione, cefinenoxime,aprindine, etomidate, mitiglinide, benidipine, levosimendan andzonisamide, or salts or prodrugs or derivatives or sustained releaseformulations thereof, for simultaneous, separate or sequentialadministration.

Most preferred drug combinations comprise 2, 3, 4 or 5 distinct drugs,even more preferably 2 or 3. Furthermore, the above drug combinationsmay also be used in further combination with additional drugs ortreatments presently used for AD.

The invention also relates to a method of treating Alzheimer's diseaseor a related disorder, the method comprising simultaneously, separatelyor sequentially administering to a subject in need thereof a drugcombination as disclosed above.

A further object of this invention is a method of treating Alzheimer'sdisease or a related disorder, the method comprising simultaneously,separately or sequentially administering to a subject in need thereof adrug combination that modulates synapse function and/or a drug thatmodulates angiogenesis and/or a drug that modulates cell stressresponse.

A further object of the invention resides in a method of producingdrug(s) for treating Alzheimer's disease or a related disorder, themethod comprising a step of testing candidate drug(s) for activity onsynapse function and angiogenesis and cellular stress response andselecting candidate drug(s) that ameliorate(s) synapse function,attenuate(s) angiogenic dysregulation and modulate(s) cellular stressresponse.

The invention further relates to a method of producing a composition fortreating Alzheimer's disease or a related disorder, the methodcomprising preparing a combination of a drug that modulates synapsefunction and/or a drug that attenuates angiogenic dysregulation and/or adrug that modulates cell stress response, for simultaneous, separate orsequential administration to a subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B: Effect of selected drugs on neurites outgrowth inbeta-amyloid intoxicated rat primary cortical neuron culture.

: p<0.01;

: p<0.00001: significantly different from vehicle. *:p<0.05;****:p<0.0001: significantly different from Abeta25-35. BilateralStudent's t test. A β₂₅₋₃₅ 20 μM produces a significant intoxication,above 25%, compared to vehicle-treated neurons. This intoxication issignificantly prevented by either Acamprosate (FIG. 1A) or Zonisamide(FIG. 1B).

FIG. 2: Effect of Phenformin on neurites outgrowth in beta-amyloidintoxicated rat primary cortical neuron culture.

:p<0.01:significantly different from vehicle. **:p<0.001: significantlydifferent from Aβ₂₅₋₃₅ Bilateral Student's t test. Aβ₂₅₋₃₅ 20 μMproduces a significant intoxication, above 25%, compared tovehicle-treated neurons. This intoxication is significantly prevented byPhenformin.

FIGS. 3A-3D: Protective effect of selected drugs against beta-amyloidpeptide toxicity on LDH release from rat endothelial cerebral cells.

: p<0.05: significantly different from vehicle.**:p<0.01; ***:p<0.0001;****:p<0.00001: significantly different from Aβ₂₅₋₃₅. BilateralStudent's t test. A β₂₅₋₃₅ 30 μM produces a moderate but significantintoxication (FIG. 3A to D). This intoxication is significantlyprevented by Leflunomide (FIG. 3A), Terbinafine (FIG. 3B), Sulfisoxazole(FIG. 3C) or Baclofen (−) (FIG. 3D). Furthermore, Leflunomide andTerbinafine not only prevent amyloid deleterious effect, but alsodecrease spontaneous cell death in the culture medium.

FIGS. 4A-4B: Effect of selected drugs on NGF-differentiated PC12viability after beta-amyloid intoxicated intoxication.

: p<0.00001: significantly different from vehicle. **:p<0.01;***:p<0.0001: significantly different from Abeta25-35. BilateralStudent's t test. Abeta25-35 10 μM produces a significant intoxication,above 25%, compared to vehicle-treated neurons (FIGS. 4A and 4B). Thisintoxication is significantly prevented by Prilocaln (FIG. 4A) orAmlodipine (FIG. 4B).

FIG. 5: Effect of selected drugs on LDH release in beta-amyloidintoxicated rat primary cortical neuron culture.

: p<0.000001: significantly different from vehicle. *:p<0.05;***:p<0.001: significantly different from Aβ₂₅₋₃₅. Bilateral Student's ttest. Aβ₂₅₋₃₅ 20 μM produces a significant intoxication, above 25%,compared to vehicle-treated neurons (FIGS. 5A and B). This intoxicationis significantly prevented by either Zonisamide (FIG. 5A) orSulfisoxazole (FIG. 5B) or Leflunomide (FIG. 5C).

FIGS. 6A-6H: Effect of selected drugs pretreatment against human Aβ₁₋₄₂injury in HBMEC. FIG. 6A: Validation of the experimental model used fordrug screening: 1 hr of VEGF pre-treatment at 10 nM significantlyprotected the capillary network from this amyloid injury (+78% ofcapillary network compared to amyloid intoxication). *: p<0.05:significantly different from control (no intoxication)

: p<0.05: significantly different from Amyloid intoxication(ANOVA+Dunett Post-Hoc test). The intoxication is significantlyprevented by Sulfisoxazole, Levosimendan, Terbinafine, Baclofen,Aminocaproic acid, Sulodexide, or Fenoldopam as shown in dose-responseexperiments, respectively in FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG.6F, FIG. 6G, FIG. 6H.

: p<0.05: significantly different from the next dose *: p<0.05:significantly different from Amyloid intoxication (ANOVA+Dunett Post-Hoctest).

FIGS. 7A-7G: Effect of selected drugs pretreatment on LDH release inhuman Aβ₁₋₄₂ toxicity on rat primary cortical cells. (A) validation ofthe experimental model used for drug screening: 1 hr of BDNF (50 ng/ml)pre-treatment significantly protected the neurons from this amyloidinjury (−62%), which is considered as a positive control forneuroprotection. *:p<0.05: significantly different from control (nointoxication)

: p<0.05: significantly different from Amyloid intoxication(ANOVA+Dunett Post-Hoc test). For all experiments, Aβ₁₋₄₂ produces asignificant intoxication compared to vehicle-treated neurons. Theintoxication is significantly prevented by Baclofen (−86%) (B),Sulfisoxazole (−42%) (C), Levosimendan (−133%) (D), Etomidate (−50%)(E), Carbenoxolone (−39%) (F), and by Cinnarizine (−50%) (G), For allexperiments,

: p<0.05:significantly different from Aβ₁₋₄₂ intoxication (ANOVA+DunettPost-Hoc test).

FIGS. 8A-8C: Effect of Sulfisoxazole and Levosimendan combinationtherapy on the total length of capillary network in beta-amyloidintoxicated HBMEC cultures.

: p<0.05, significantly different from Aβ₁₋₄₂. *:p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofSulfisoxazole and Levosimendan (A) whereas, at those concentrations,Levosimendan (B) and Sulfisoxazole (C) alone have no significant effecton intoxication.

FIGS. 9A-9C: Effect of Sulfisoxazole and Terbinafine combination therapyon the total length of capillary network in beta-amyloid intoxicatedHBMEC cultures.

p<0.05, significantly different from Aβ₁₋₄₂. *: p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofSulfisoxazole and Levosimendan (A) whereas, at those concentrations,Sulfisoxazole (B) and Terbinafine (C) alone have no significant effecton intoxication.

FIGS. 10A-10C: Effect of Baclofen and Levosimendan combination therapyon the total length of capillary network in beta-amyloid intoxicatedHBMEC cultures.

:p<0.05, significantly different from Aβ₁₋₄₂. *:p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination of Baclofenand Levosimendan (A) whereas, at those concentrations, Levosimendan (B)and Baclofen (C) alone have no significant effect on intoxication.

FIGS. 11A-11C: Effect of Terbinafine and Aminocaproic acid combinationtherapy on the total length of capillary network in beta-amyloidintoxicated HBMEC cultures.

:p<0.05, significantly different from Aβ₁₋₄₂. *:p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofTerbinafine and Aminocaproic acid (A) whereas, at those concentrations,Aminocaproic acid (B) and Terbinafine (C) alone have no significanteffect on intoxication.

FIGS. 12A-12C: Effect of Aminocaproic acid and Levosimendan combinationon the total length of capillary network in beta-amyloid intoxicatedHBMEC cultures.

:p<0.05, significantly different from Aβ₁₋₄₂. *:p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofLevosimendan and Aminocaproic acid (A) whereas, at those concentrations,Aminocaproic acid (B) and Levosimendan (C) alone have no significanteffect on intoxication.

FIGS. 13A-13C: Effect of Terbinafine and Levosimendan combinationtherapy on the total length of capillary network in beta-amyloidintoxicated HBMEC cultures.

: p<0.05, significantly different from Aβ₁₋₄₂. *:p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofTerbinafine and Levosimendan (A) whereas, at those concentrations,Terbinafine (B) and Levosimendan (C) alone have no significant effect onintoxication.

FIG. 14: Effect of Carbamazine (CARB) and Acamprosate (ACP) combinationtherapy against human Aβ₁₋₄₂ toxicity on LDH release from rat primarycortical cells. *: p<0.05: significantly different from control (nointoxication).

p<0.005: significantly different from Amyloid intoxication (ANOVA+DunettPost-Hoc test). The aggregated human amyloid peptide (Aβ₁₋₄₂ 2.5 μM)produces a significant intoxication. This intoxication is significantlyprevented by the combination of Carbamazine (0.32 nM) and Acamprosate(0.32 nM) whereas, at those concentrations, Carbamazine and Acamprosatealone have no significant effect on intoxication.

FIGS. 15A-15C: Effect of Sulfisoxazole and Sulodexide combinationtherapy on the total length of capillary network in beta-amyloidintoxicated HBMEC cultures.

: p<0.05, significantly different from Aβ₁₋₄₂. *: p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication. This intoxication is significantly prevented by thecombination of Sulfisoxazole (1.36 nM) and Sulodexide (0.002 LRU/mL) (A)whereas, at those concentrations, Sulfisoxazole (B) and Sulodexide (C)alone have no significant effect on intoxication.

FIG. 16: Effect of Aminocaproic acid and Torasemide combination therapyon the total length of capillary network in beta-amyloid intoxicatedHBMEC cultures.

: p<0.05, significantly different from Aβ₁₋₄₂. *: p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication. This intoxication is significantly prevented by thecombination of Aminocaproic acid (160 nM) and Torasemide (400 nM).

FIG. 17: Effect of Torasemide and Levosimendan combination therapy onthe total length of capillary network in beta-amyloid intoxicated HBMECcultures.

: p<0.05, significantly different from Aβ₁₋₄₂. *: p<0.05, significantlydifferent from vehicle (ANOVA+Bunett Post-Hoc test). The aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication. This intoxication is significantly prevented by thecombination of Torasemide (400 nM) and Levosimendan (1.6 nM).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new therapeutic approaches for treatingAD or related disorders. The invention discloses novel use of drugs ordrug combinations which allow an effective correction of such diseasesand may be used for patient treatment.

The term “AD related disorder” includes senile dementia of AD type(SDAT), Parkinson's disease, Lewis body dementia, vascular dementia,mild cognitive impairment (MCI), age-associated memory impairment (AAMI)and problem associated with ageing, post-encephalitic Parkinsonism,Amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and Downsyndrome.

As used herein, “treatment” of a disorder includes the therapy,prevention, prophylaxis, retardation or reduction of symptoms provokedby the disorder. The term treatment includes in particular the controlof disease progression and associated symptoms.

The term “ameliorate”, as it refers to synapse function, includes anyincrease in the synapse function as compared to the existing function inthe subject. Such amelioration may include a restoration, i.e., tonormal levels, or lower increase, which are still sufficient to improvethe patient condition. Such amelioration can be evaluated or verifiedusing known biological tests, such as described in the experimentalsection.

The term “increase”, as it refers to angiogenesis, includes any increasein the angiogenesis as compared to the existing level in the subject.Such amelioration may include a restoration, i.e., to normal levels, orlower increase, which are still sufficient to improve the patientcondition. Such an increase can be evaluated or verified using knownbiological tests, such as described in the experimental section.

The term “inhibit”, as it refers to cell stress response (“CSR”),includes any reduction in the CSR as compared to the existing activityin the subject. Such reduction may include a partial diminution, e.g.,from 5-20%, which is sufficient to improve the patient condition, aswell as more substantial reductions, e.g., from 20-50% or more completeinhibition, e.g., above 50%. The inhibition can be evaluated or verifiedusing known biological tests, such as described in the experimentalsection.

Also, the designation of specific compounds within the context of thisinvention is meant to include not only the specifically named molecules,but also any pharmaceutically acceptable salt, hydrate, ester, ether,isomers, racemate, conjugates, or pro-drugs thereof of any purity.

The term “combination or combinatorial treating/therapy” designates atreatment wherein at least two or more drugs are co-administered to asubject to cause a biological effect. In a combined therapy according tothis invention, the at least two drugs may be administered together orseparately, at the same time or sequentially. Also, the at least twodrugs may be administered through different routes and protocols. As aresult, although they may be formulated together, the drugs of acombination may also be formulated separately.

As discussed above, the invention relates to compositions and methodsfor treating Alzheimer's disease or a related disorder in a subject inneed thereof, using particular drugs or drug combinations thatameliorate synapse function and/or increases angiogenesis and/orinhibits cell stress response.

By a comprehensive integration of experimental data covering results ofcell biology studies, expression profiling experiments and geneticassociation studies, describing different aspects of Alzheimer's diseaseand links existing in cellular signalling and functional pathways, theinventors have found that synapse function, angiogenesis and cell stressresponse represent important mechanisms which are altered in subjectshaving AD. By further experimental investigations, the inventors haveselected drugs or drug combinations which effectively alter thesepathways and which effectively improve AD, as illustrated in theexamples. These drugs and combinations thus represent novel approachesfor treating AD and related disorders.

Genes located in said functional networks and implicated in Alzheimer'sdisease were selected by the following criteria:

-   -   (1)—direct interaction with the genes causatively responsible        for familial cases of Alzheimer's disease (APP, ApoE,        presenilins, tau protein),    -   (2)—functional partners of the genes selected by the criterion        (1),    -   (3)—nearest functional partners of the genes selected by the        criterion (2).

Through this process, the inventors have established that the networksresponsible for synapse function, angiogenesis and cell stress responseare major functional networks affected in Alzheimer's disease.

The inventors have more specifically established that the synaptic lossis a functionally-relevant hallmark of Alzheimer's disease, whichultimately leads to progressive cognitive decline, memory loss anddementia. Importantly, synaptic loss correlates better with cognitivedeficit characterized Alzheimer's pathology, compared to otherAD-specific cellular lesion markers manifested in development ofneurofibrillary tangles or deposition of amyloid plaques. Consequently,synapse organization and synaptic plasticity represent an importanttarget for therapeutic interventions in the context of Alzheimer'sdisease.

APP protein is axonally transported and processed in presynapticterminals, leading to high accumulation of Abeta at synapses. Oligomersof Abeta42 as well as amyloid plaques themselves are important forinhibiting long-term potentiation and are primarily responsible formemory impairment in AD patients.

Our data integration procedure revealed a group of genes, which areimplicated in synaptic distortion in AD and which can be formallyseparated into three main functional groups: proteins participating inorganization of post-synaptic density (“PSD”) and correct nerve signaltransmission at post-synaptic membrane; proteins assuringneurotransmitter release; and proteins involved in axon growth anddevelopmental maturation of synaptic machinery.

In a particular embodiment, the present invention thus recognizes thatit is important, for efficient treatment of AD, to ameliorate theactivity of proteins involved in post-synaptic density.

Among genes identified by our analysis, the DLG2 gene, which encodesMAGUK family protein and creates an interface between clusteredmembrane-bound receptors, cell-adhesion molecules and actin-basedcytoskeleton, represents a particular interest (17-18). The inventorshave identified a large group of ionotrophic/metabotrophic glutamate andgrowth factor receptors, which interact directly with the DLG2 proteinor DLG2/PSD95 proteins complex at excitatory synapses and which can betherefore recognized as therapeutic targets for treating Alzheimer'sdisease.

In another particular embodiment, the present invention thus alsorecognizes that it is important, for efficient treatment of AD, toameliorate the activity of proteins involved in the regulation ofneurotransmitter release at the pre-synaptic membrane.

The release of neurotransmitters at a restricted and highly specializedactive zone of the presynaptic plasma membrane is triggered by actionpotential and is controlled by combined actions of voltage-dependent,calcium Ca_(v) channels, MaxiK/BK channels (potassium large conductancecalcium-activated channels) and cGMP-dependent PRKG protein kinases, allof which are tightly associated,—as demonstrated by our analysis,—withdevelopment of Alzheimer's disease. In addition to these functionalmodules implicated in neurotransmitter release, the inventors havedefined another group of proteins, linked to dysregulation of synapticneurotransmission in course of Alzheimer's disease, which areresponsible for maturation, docking and fusion of synaptic vesicles (forinstance, STX2, STXBP6, BIN1, RAB3B, UNC13C and RIMS1/2 scaffoldingproteins). These functional pathways were therefore prioritized asappropriate therapeutic targets for treatment of Alzheimer's disease.

In another particular embodiment, the present invention furtherrecognizes that it is important, for efficient treatment of AD, toameliorate the activity of proteins involved in the regulation of axongrowth and guidance.

Proteins participating in regulation of axon growth and guidance allowneuronal precursor cells and axons to migrate toward proper destinationsto ensure correct location and connectivity; they are also involved indevelopmental maturation of newly established synapses as well asdegradation of axons and synopsis in AD disease. These processes play afundamental role for execution of cognitive functions and seem to beextremely vulnerable to toxic effect of Abeta depositions.

Consecutive steps of axon growth and guidance are tightly controlled bycombined actions of extracellular or membrane-tethered Netrins,Semaphorins, Ephrins, DLL and Slits molecules and their respectivefunctional receptors, most of which were revealed by our data miningapproach. Functional outcomes of activation of most of axon growthreceptors are tightly connected with their ability to differentiallymodulate activity of small GTPases RhoA, Rac1 and Cdc42, with the RhoAGTPase being mainly responsible for neurite retraction and growth conecollapse (19). These signalling pathways have been recognized aspertinent therapeutic targets for treatment of Alzheimer's disease.

Thus, the present invention recognizes that it is important, forefficient treatment of AD, to ameliorate synapse function altered inAlzheimer's disease and other neurogenerative disorders, by modulatingtarget genes and protein described above.

Through data mining process, the inventors have also established thatthe network responsible for angiogenesis represents another majorfunctional network affected in Alzheimer's disease.

Angiogenesis plays a fundamental role in ensuring a tissue homeostasisand in adaptive responses to environmental and physiological challengessuch as hypoxia or wound healing; its dysfunction contributes to thepathogenesis of numerous and heterogeneous pathologies varying fromcardiovascular complications to tumour's growth and metastasis.

Although Alzheimer's disease is traditionally considered as aneurodegenerative condition accompanied by collateral vascularpathology, our analysis allow re-evaluation of the pathogenic impact ofthe vascular deregulation and attribute an important and probablycausative role to angiogenic pathways in aetiology of this disease. Theinventors have found that genes regulating angiogenesis are extremelyenriched in signalling networks implicated in Alzheimer's disease. Thisconclusion has deep consequences for prevention and curing ofAlzheimer's disease and provides new guidelines for combinatorialtreatment of this complex neurodegenerative disorder.

Among signalling pathways tightly implicated in vascular remodellingassociated with Alzheimer's disease, several functional modules mediatedby VEGFR1, ErbB4, Notch, DCC, CD44, ephrin receptors and cadherins havebeen identified.

As revealed by our data mining approach, other target proteins,potentially involved in development of vascular defects manifested incourse of Alzheimer's disease, include IL20Rα, LEPTR, NRP1 and NRP2, andendothelin EDNRA receptors, proteins participating in organization andremodelling of extracellular matrix (THBS2, LAMA1, COL4A2, ADAMTS12 andADAM10) or proteins (for instance, TLL2) playing an important role infunctional processing of well-known angiogenic modulators such asprolactin, growth hormone, and placental lactogen (20).

Further, we have also discovered that several genes, associated withAlzheimer's disease, represent upstream modulators and down-streameffectors of the AMP-activated kinases, important regulators of vascularsystem (for instance, leptin and CNTF receptors, trombin signallingpathway, CAMKK2β and LKB1 kinases) (21-24). This finding allowed us todefine AMPK-mediated signalling network as a reasonable therapeutictarget for treatment of Alzheimer's disease.

Phosphatidic acid (PA), lysophosphatidic acid (LPA), and sphingosine1-phosphate (S1P) are natural phospholipids that possess potentsignaling properties. Notably, these phospholipid growth factors displaydivergent effects on angiogenic potential of endothelial cells (25).Using our data mining approach, we identified a large number of genes,involved in LPA metabolism or modulated by LPA signaling and potentiallylinked to progression of Alzheimer's disease (MTR, MAT2B, CUBN, ATP10A,THEM2, PITPNC1, ENPPG, SGPP2, AGPAT, DGKH, DGKB, MGST2, PLD2, and DRD2).Therefore, we concluded that this signaling network represents asuitable therapeutic target for treatment of Alzheimer's disease.

The present invention also emphasizes the importance of increasingangiogenesis altered in Alzheimer's disease and other neurogenerativedisorders, by modulating target genes and protein described above.

Finally, we have established that the network responsible for cellstress response is the 3rd major functional network affected inAlzheimer's disease.

We have more specifically established that cell stress response is afunctionally-relevant hallmark of Alzheimer's disease. As discussedbelow, the inventors have identified three families of proteins, withinthe cell stress response network, which are functionally relevant to thegenesis and control of Alzheimer's disease, and represent valuabletargets for combination therapies. These groups of proteins are, morespecifically, proteins participating in calcium homeostasis, in proteinfolding, and in execution of apoptosis.

In a particular embodiment, the present invention more specificallyrelates to compositions and methods using a drug combination thatmodulates the activity of a protein involved in calcium homeostasis.

Calcium, one of the most important intracellular messengers, mediates apleiotropy of cellular processes in both neuronal and endothelial cells,including synaptic plasticity, angiogenesis and apoptosis. Intracellularcalcium level is precisely regulated by cooperative action of a seriesof calcium permeable channels, calcium pumps and calcium exchangers inplasma membrane and endoplasmatic reticulum (26-27). We have identifieda network of genes implicated in calcium homeostasis pathway, whosefunction could be modified by mutant presenilin proteins or by toxicβ-amyloid in course of Alzheimer's disease. Among them, IP3R (ITPR1) andRYR3 receptors, ATP2A3 (SERCA3 Ca2+ ATPase) regulating calciumhomeostasis on the level of ER, plasma membrane ATPase ATP2B1, extrudingcalcium ions from eukaryotic cells against concentration gradients, andvoltage-gated Na⁺ channels represent particular interest as potentialtherapeutic targets for treatment of Alzheimer's disease.

In another particular embodiment, the present invention morespecifically relates to compositions and methods using a drugcombination that modulates the activity of a protein involved in proteinfolding or aggregation.

Protein aggregation is a central cytopathological phenomenon in AD. Twomajor cellular hallmarks of Alzheimer's disease are manifested indevelopment of neurofibrillary tangles (NFTs) and deposition of amyloidplaques, composed of aggregated hyperphosphorylated tau protein and Aβfragments of APP protein respectively. Another protein prone toaggregation—α-synuclein, recognized as rather specific hallmark ofParkinson Disease, can be nevertheless detected in amyloid plaques inmost cases of sporadic and familial forms of Alzheimer's disease.

We have determined several genes implicated in modulation of folding,posttranslational modification and processing of every major constituentof Alzheimer's disease-associated protein's aggregations as pertinenttherapeutic targets for treatment of Alzheimer's disease—for instance,APBA1 and APBA2BP proteins that interact with APP and regulate itsstability and functions, or PARK2 ubiquitin-protein ligase that isimplicated in clearance of α-synuclein (28). As well, the GSK-3β kinasemight play a particularly important role in pathogenesis of proteinmisfolding in course of Alzheimer's disease. This conclusion isre-enforced by our finding that a few signalling modules regulatingGSK-3β kinase activity and its interaction with tau protein—WWOX (29),hyaluronan CD44 receptor, Wnt receptors Fz2/ROR2 and insulinreceptor/PTPRG phosphatase complex (30)—are associated with progressionof Alzheimer's disease.

In a further particular embodiment, the present invention relates tocompositions and methods using a drug combination that inhibitsapoptosis that is recognized as a major cellular mechanism responsiblefor cellular loss in Alzheimer's disease.

As identified by our analysis, apoptosis in the case of Alzheimer'sdisease, most likely, is executed via canonical p53-dependent pathways.

The p53 protein can be regulated through post-translationalmodifications and through interactions with positive and negativeregulatory factors. We have identified several such regulatoryproteins—WWOX, MDM1, HIPK2 and PML—confirming the proposal about thepivotal role of the p53 protein in cell death execution in Alzheimer'sdisease (31-33).

Among the receptor systems that could be directly and specificallyimplicated in induction of apoptosis in context of Alzheimer's disease,UNC5C (Unc-5 Homolog C) and DCC (Deleted in Colorectal Carcinoma) netrinreceptors, involving in axon guidance and angiogenesis, representparticular interest. These receptors are designated putative conditionaltumor suppressors, since they behave as netrin-dependent receptorsinducing apoptosis in the absence of their ligand (34). Binding ofnetrin-1 to these receptors inhibits p53-dependent apoptosis, while p53is directly involved in transcriptional regulation of netrin-1 and itsreceptors (33). Additionally, the DCC receptor is known to be processedby presenilin, indicating its important role in development ofAlzheimer's disease (35). Thus, our data mining suggests that netrinreceptors-dependent and p53-mediated programmed cell death could be oneof the specific pro-apoptotic pathways implicated in pathological cellloss in context of Alzheimer's disease, in addition to rather unspecificpro-apoptotic programs stimulated by disrupted calcium homeostasis andexcessive ROS production.

In a particular embodiment, the present invention more specificallyrelates to compositions and methods using a drug combination thatinhibits the activity of at least two distinct proteins involved incalcium homeostasis, in protein folding, and in execution of apoptosis.

In a preferred embodiment, the present invention proposes novelcompositions, which can be used to inhibit cell stress response inducedin Alzheimer's disease and other neurogenerative disorders, bymodulating target genes and protein described above.

As discussed above, the invention relates to compositions and methodsfor treating Alzheimer's disease or a related disorder in a subject inneed thereof, using a combination of drugs that ameliorate synapsefunction and/or increases angiogenesis and/or inhibits cell stressresponse.

More specifically, the inventors have selected and tested a number ofdrugs or drug combinations which alter one or, preferably, all of theabove described pathways. As disclosed in the examples, these drugcombinations have a strong effect on Alzheimer's disease and representnew therapeutic approaches of the pathology. These drug combinations areparticularly advantageous because they affect different pathways andthus are more effective. Also, because of their efficacy and mode ofaction, the drug combinations can be used at low dosages, which is afurther very substantial advantage. The most preferred drugs are listedin Table 1 below.

TABLE 1 DRUG NAME CAS NUMBER Acamprosate 77337-76-9 Ambrisentan177036-94-1 Aminocaproic Acid 60-32-2 Amlodipine 88150-42-9 Amobarbital57-43-2 Aprindine 37640-71-4 Argatroban 74863-84-6 Baclofen 1134-47-0Benidipine 105979-17-7 Carbamazepine 298-46-4 Carbamazine 90-89-1Carbenoxolone 5697-56-3 Cefmenoxime 65085-01-0 Cefotetan 69712-56-7Ciclopirox 29342-05-0 Cilostazol 73963-72-1 Cinacalcet 226256-56-0Cinnarizine 298-57-7 Clopidogrel 113665-84-2 Dyphylline 479-18-5Enprofylline 41078-02-8 Eplerenone 107724-20-9 Eprosartan 133040-01-4Erythrityl tetranitrate 7297-25-8 Etomidate 33125-97-2 Fenoldopam67227-57-0 Leflunomide 75706-12-6 Lercanidipine 100427-26-7 Levosimendan141505-33-1 Mepacrine 83-89-6 Methimazole 60-56-0 Methyclothiazide135-07-9 Mitiglinide 145375-43-5 Moxifloxacin 354812-41-2 Oxtriphylline4499-40-5 Paramethadione 115-67-3 Phenformin 114-86-3 Prilocaine721-50-6 Rifabutin 72559-06-9 Risedronate 105462-24-6 Sulfisoxazole127-69-5 Sulodexide 57821-29-1 Tadalafil 171596-29-5 Terbinafine91161-71-6 Torasemide 56211-40-6; 72810-59-4 Zonisamide 68291-97-4

In this regard, a preferred object of this invention relates tocompositions comprising a combination of at least two compounds chosenfrom the group consisting of aminocaproic acid, acamprosate, amlodipine,argatroban, baclofen, cilostazol, cinacalcet, clopidogrel, dyphylline,fenoldopam, leflunomide, mepacrine, methimazole, phenformin, prilocalne,rifabutin, sulfisoxazole, tadalafil, terbinafine, torasemide,cinnarizine, ciclopirox, eplerenone, carbenoxolone, sulodexide,carbamazine, amobarbital, cefotetan, erythrityl tetranitrate,methyclothiazide, risedronate, enprofylline, oxtriphylline,paramethadione, cefinenoxime, aprindine, etomidate, mitiglinide,benidipine, levosimendan and zonisamide, or salts or prodrugs orderivatives of any purity or sustained release formulations thereof, forsimultaneous, separate or sequential administration.

In a particular embodiment, the invention relates to compositionscomprising at least one compound chosen from the group consisting ofaminocaproic acid, cinnarizine, ciclopirox, eplerenone, carbenoxolone,sulodexide, carbamazine, amobarbital, cefotetan, erythrityltetranitrate, methyclothiazide, risedronate, enprofylline,oxtriphylline, paramethadione, cefinenoxime, aprindine, etomidate,mitiglinide, benidipine and levosimendan, or salt(s) or prodrug(s) orderivative(s) or sustained release formulation(s) thereof, incombination with at least one compound chosen from the group consistingof acamprosate, amlodipine, argatroban, baclofen, cilostazol,cinacalcet, clopidogrel, dyphylline, fenoldopam, leflunomide, mepacrine,methimazole, phenformin, prilocalne, rifabutin, sulfisoxazole,tadalafil, terbinafine, torasemide and zonisamide, or salt(s) orprodrug(s or derivative(s) or sustained release formulation(s) thereof,for simultaneous, separate or sequential administration.

As disclosed in the examples, combination therapies using at least 2 ofthe above-listed drugs lead to an efficient correction of Alzheimer'sdisease.

Therapy according to the invention may be performed alone or as drugcombination.

In a preferred embodiment, the drugs of the invention are used incombination(s) for combined, separate or sequential administration, inorder to provide the most effective effect. In this respect, thecompositions of treating Alzheimer's disease according to the inventionuse drug(s) that ameliorate synapse function and drug(s) that attenuateangiogenesis and/or drug(s) that inhibit cell stress response.

More specifically, the compositions according to the invention, for usein the treatment of Alzheimer's disease or a related disorder, may beselected from compositions comprising at least one of the followingcombinations of drugs:

-   -   a modulator of AMPK (preferably, phenformin) and an inhibitor of        sodium channel SCN1A and an activator of BK channels        (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide),    -   a modulator of AMPK (preferably, phenformin) and a modulator of        GABAergic and glutamatergic receptors activity (preferably        selected from acamprosate, etomidate and aprindine),    -   a modulator of AMPK (preferably, phenformin) and an antagonist        of EDNRA endothelin receptor (preferably, sulfisoxazole),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and a modulator of RYR3 ryanodine        receptor (preferably, prilocalne),    -   a modulator of GABBR2 receptor (preferably, baclofen) and a        modulator of RHOA (preferably selected from terbinafine and        risedronate),    -   a modulator of GABBR2 receptor (preferably, baclofen) and an        antagonist of EDNRA endothelin receptor (preferably,        sulfisoxazole),    -   a modulator of GABBR2 receptor (preferably, baclofen) and an        inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide),    -   a modulator of GABBR2 receptor (preferably, baclofen) and a        modulator of HAS1-3 hyaluronan synthases (preferably,        leflunomide),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and a modulator of adenosine        receptors ADORA1/2/3 (preferably, dyphylline),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and an antagonist of EDNRA        endothelin receptor (preferably, sulfisoxazole),    -   a modulator of RHOA (preferably selected from terbinafine and        risedronate) and an antagonist of EDNRA endothelin receptor        (preferably, sulfisoxazole),    -   a modulator of RHOA (preferably selected from terbinafine and        risedronate) and an inhibitor of phospholipases PLA1A and PLA2        (preferably, mepacrine),    -   a modulator of RHOA (preferably selected from terbinafine and        risedronate) and a modulator of GABAergic and glutamatergic        receptors activity (preferably selected from acamprosate,        etomidate and aprindine),    -   a modulator of RHOA (preferably selected from terbinafine and        risedronate) and a chemical chaperon (preferably, rifabutin),    -   a modulator of AMPK (preferably, phenformin) and an inhibitor of        PDEllA and PDE4A, PDE5A phosphodiesterases (preferably selected        from tadalafil, enprofylline and oxtriphylline),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and a modulator of trombin        receptor F2R signalling (preferably, argatroban and        cefinenoxime),    -   a modulator of AMPK (preferably, phenformin) and a modulator of        purinergic receptors P2RY1 and P2RY12 (preferably, clopidogrel),    -   a modulator of GABAergic and glutamatergic receptors activity        (preferably selected from acamprosate, etomidate and aprindine)        and a modulator of CASR (preferably, cinacalcet),    -   an antagonist of EDNRA endothelin receptor (preferably,        sulfisoxazole) and a modulator of CASR (preferably, cinacalcet),    -   a modulator of RHOA (preferably selected from terbinafine and        risedronate) and a modulator of trombin receptor F2R signalling        (preferably selected from argatroban and cefinenoxime),    -   a modulator of GABBR2 receptor (preferably, baclofen) and a        modulator of purinergic receptors P2RY1 and P2RY12 (preferably,        clopidogrel),    -   a modulator of RHOA (preferably selected from terbinafine and        risedronate) and a modulator of purinergic receptors P2RY1 and        P2RY12 (preferably, clopidogrel),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and an antagonist of        voltage-gated calcium CACNA channels (preferably selected from        cinnarizine, benidipine, paramethadione and amlodipine),    -   a modulator of GABAergic and glutamatergic receptors activity        (preferably selected from acamprosate, etomidate and aprindine)        and an antagonist of voltage-gated calcium CACNA channels        (preferably selected from cinnarizine, benidipine,        paramethadione and amlodipine),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and a modulator of HIF1A        signalling (preferably, ciclopirox),    -   a modulator of GABAergic and glutamatergic receptors (preferably        selected from acamprosate, etomidate and aprindine) and a        modulator of HIF1A signalling (preferably, ciclopirox),    -   an antagonist of EDNRA endothelin receptor (preferably,        sulfisoxazole) and a modulator of oxidative phosphorylation        (preferably selected from amobarbital and methimazole),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and a modulator of oxidative        phosphorylation (preferably selected from amobarbital and        methimazole),    -   an antagonist of EDNRA endothelin receptor (preferably,        sulfisoxazole) and a modulator of vitamin K metabolism        (preferably, cefotetan),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and a modulator of vitamin K        metabolism (preferably, cefotetan),    -   a modulator of GABAergic and glutamatergic receptors activity        (preferably selected from acamprosate, etomidate and aprindine)        and a modulator of PRKG1 (preferably, erythrityl tetranitrate),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) or a modulator of BK channels        (preferably, methyclothiazide) and a modulator of PRKG1        (preferably, erythrityl tetranitrate),    -   an antagonist of EDNRA endothelin receptor (preferably,        sulfisoxazole) and a modulator of PRKG1 (preferably, erythrityl        tetranitrate),    -   a modulator of KCNJ11 (preferably selected from mitiglinide and        levosimendan) and a modulator of PRKG1 (preferably, erythrityl        tetranitrate),    -   a modulator of KCNJ11 (preferably selected from mitiglinide and        levosimendan) and—an inhibitor of sodium channel SCN1A and an        activator of BK channels (preferably, zonisamide) or a modulator        of BK channels (preferably, methyclothiazide),    -   a modulator of KCNJ11 (preferably selected from mitiglinide and        levosimendan) and—a modulator of RHOA (preferably selected from        terbinafine and risedronate).

In the most preferred embodiment, the invention relates to anycombination of compounds selected from aminocaproic acid, acamprosate,amlodipine, argatroban, baclofen, cilostazol, cinacalcet, clopidogrel,dyphylline, fenoldopam, leflunomide, mepacrine, methimazole, phenformin,prilocalne, rifabutin, sulfisoxazole, tadalafil, terbinafine,torasemide, cinnarizine, ciclopirox, eplerenone, carbenoxolone,sulodexide, carbamazine, amobarbital, cefotetan, erythrityltetranitrate, methyclothiazide, risedronate, enprofylline,oxtriphylline, paramethadione, cefinenoxime, aprindine, etomidate,mitiglinide, benidipine, levosimendan and zonisamide, or salts orprodrugs or derivatives or sustained release formulations thereof, foruse in the treatment of Alzheimer's disease or a related disorder.

In a particular embodiment, a composition of the invention, for use inthe treatment of Alzheimer's disease or a related disorder, comprises atleast one compound chosen from the group consisting of aminocaproicacid, cinnarizine, ciclopirox, eplerenone, carbenoxolone, sulodexide,carbamazine, amobarbital, cefotetan, erythrityl tetranitrate,methyclothiazide, risedronate, enprofylline, oxtriphylline,paramethadione, cefinenoxime, aprindine, etomidate, mitiglinide,benidipine and levosimendan, or salt(s) or prodrug(s) or derivative(s)or sustained release formulation(s) thereof, in combination with atleast one compound chosen from the group consisting of acamprosate,amlodipine, argatroban, baclofen, cilostazol, cinacalcet, clopidogrel,dyphylline, fenoldopam, leflunomide, mepacrine, methimazole, phenformin,prilocalne, rifabutin, sulfisoxazole, tadalafil, terbinafine, torasemideand zonisamide, or salt(s) or prodrug(s) or derivative(s) or sustainedrelease formulation(s) thereof.

Another particularly preferred embodiment of the invention relates to acomposition for treating Alzheimer's disease (AD) or a related disorderin a subject in need thereof, comprising at least aminocaproic acid, orsalt(s) or prodrug(s) or derivative(s) or sustained releaseformulation(s) thereof. In a particular embodiment, aminocaproic acid isused in combination with at least one additional compound preferablyselected from acamprosate, amlodipine, argatroban, baclofen, cilostazol,cinacalcet, clopidogrel, dyphylline, fenoldopam, leflunomide, mepacrine,methimazole, phenformin, prilocalne, rifabutin, sulfisoxazole,tadalafil, terbinafine, torasemide, cinnarizine, ciclopirox, eplerenone,carbenoxolone, sulodexide, carbamazine, amobarbital, cefotetan,erythrityl tetranitrate, methyclothiazide, risedronate, enprofylline,oxtriphylline, paramethadione, cefinenoxime, aprindine, etomidate,mitiglinide, benidipine, levosimendan, and zonisamide, or salts orprodrugs or derivatives or sustained release formulations thereof, forcombined, separate or sequential administration.

A preferred composition of the invention comprises aminocaproic acid, orsalt(s) or prodrug(s) or derivative(s) or sustained releaseformulation(s) thereof, and at least one additional compound selectedfrom baclofen, sulfisoxazole, terbinafine, torasemide, and levosimendan,or salts or prodrugs or derivatives or sustained release formulationsthereof, for combined, separate or sequential administration. Such acomposition per se also represents a particular object of the invention.

The invention also relates to a method of treating Alzheimer's disease(AD) or a related disorder in a subject in need thereof, comprisingadministering to the subject an effective amount of aminocaproic acid,or salt(s) or prodrug(s) or derivative(s) or sustained releaseformulation(s) thereof, preferably in combination as disclosed above.

Another particularly preferred embodiment of the invention relates to acomposition for treating Alzheimer's disease (AD) or a related disorderin a subject in need thereof, comprising at least levosimendan, orsalt(s) or prodrug(s) or derivative(s) or sustained releaseformulation(s) thereof. In a particular embodiment, levosimendan is usedin combination with at least one additional compound preferably selectedfrom aminocaproic acid, acamprosate, amlodipine, argatroban, baclofen,cilostazol, cinacalcet, clopidogrel, dyphylline, fenoldopam,leflunomide, mepacrine, methimazole, phenformin, prilocalne, rifabutin,sulfisoxazole, tadalafil, terbinafine, torasemide, cinnarizine,ciclopirox, eplerenone, carbenoxolone, sulodexide, carbamazine,amobarbital, cefotetan, erythrityl tetranitrate, methyclothiazide,risedronate, enprofylline, oxtriphylline, paramethadione, cefinenoxime,aprindine, etomidate, mitiglinide, benidipine, and zonisamide, or saltsor prodrugs or derivatives or sustained release formulations thereof,for combined, separate or sequential administration.

A preferred composition of the invention comprises levosimendan, orsalt(s) or prodrug(s) or derivative(s) or sustained releaseformulation(s) thereof, and at least one additional compound selectedfrom aminocaproic acid, baclofen, sulfisoxazole, terbinafine, andtorasemide, or salts or prodrugs or derivatives or sustained releaseformulations thereof, for combined, separate or sequentialadministration. Such a composition per se also represents a particularobject of the invention.

The invention also relates to a method of treating Alzheimer's disease(AD) or a related disorder in a subject in need thereof, comprisingadministering to the subject an effective amount of levosimendan, orsalt(s) or prodrug(s) or derivative(s) or sustained releaseformulation(s) thereof, preferably in combination as disclosed above.

Another particularly preferred embodiment of the invention relates to acomposition for treating Alzheimer's disease (AD) or a related disorderin a subject in need thereof, the composition comprising at leastEplerenone, Carbenoxolone, Sulodexide, Cinnarizine, or carbamazine, orsalt(s) or prodrug(s) or derivative(s) or sustained releaseformulation(s) thereof.

In a particular embodiment, Eplerenone, Carbenoxolone, Sulodexide,Cinnarizine, or carbamazine, is used in combination with at least oneadditional compound preferably selected from levosimendan, aminocaproicacid, acamprosate, amlodipine, argatroban, baclofen, cilostazol,cinacalcet, clopidogrel, dyphylline, fenoldopam, leflunomide, mepacrine,methimazole, phenformin, prilocalne, rifabutin, sulfisoxazole,tadalafil, terbinafine, torasemide, cinnarizine, ciclopirox, eplerenone,carbenoxolone, sulodexide, carbamazine, amobarbital, cefotetan,erythrityl tetranitrate, methyclothiazide, risedronate, enprofylline,oxtriphylline, paramethadione, cefinenoxime, aprindine, etomidate,mitiglinide, benidipine, and zonisamide, or salts or prodrugs orderivatives or sustained release formulations thereof, for combined,separate or sequential administration.

A preferred composition of the invention comprises Eplerenone,Carbenoxolone, Sulodexide, Cinnarizine, or carbamazine, or salt(s) orprodrug(s) or derivative(s) or sustained release formulation(s) thereof,and at least one additional compound selected from levosimendan,aminocaproic acid, baclofen, sulfisoxazole, terbinafine, and torasemide,or salts or prodrugs or derivatives or sustained release formulationsthereof, for combined, separate or sequential administration. Such acomposition per se also represents a particular object of the invention.

The invention also relates to a method of treating Alzheimer's disease(AD) or a related disorder in a subject in need thereof, comprisingadministering to the subject an effective amount of Eplerenone,Carbenoxolone, Sulodexide, Cinnarizine, or carbamazine, or salt(s) orprodrug(s) or derivative(s) or sustained release formulation(s) thereof,preferably in combination as disclosed above.

More preferably, the composition of the invention, for combinatorialtreating Alzheimer's disease (AD) or a related disorder in a subject inneed thereof, comprises at least one of the following drug combinationsfor combined, separate or sequential administration:

-   -   phenformin and zonisamide,    -   phenformin and methyclothiazide,    -   phenformin and acamprosate,    -   phenformin and sulfisoxazole,    -   baclofen and aminocaproic acid,    -   baclofen and levosimendan,    -   baclofen and terbinafine,    -   baclofen and risedronate,    -   baclofen and sulfisoxazole,    -   baclofen and zonisamide,    -   baclofen and methyclothiazide,    -   baclofen and sulfisoxazole,    -   baclofen and leflunomide,    -   aminocaproic acid and sulfisoxazole,    -   aminocaproic acid and terbinafine,    -   aminocaproic acid and levosimendan,    -   levosimendan and sulfisoxazole,    -   levosimendan and terbinafine,    -   zonisamide and dyphylline,    -   methyclothiazide and dyphylline,    -   zonisamide and prilocalne,    -   methyclothiazide and prilocalne,    -   zonisamide and sulfisoxazole,    -   phenformin and clopidogrel,    -   acamprosate and cinacalcet,    -   sulfisoxazole and cinacalcet,    -   terbinafine and argatroban,    -   terbinafine and cefinenoxime,    -   baclofen and clopidogrel,    -   terbinafine and clopidogrel,    -   risedronate and clopidogrel,    -   zonisamide and cinnarizine,    -   sulodexide and sulfisoxazole,    -   torasemide and aminocaproic acid,    -   torasemide and levosimendan,    -   carbamazine and acamprosate,    -   acamprosate and erythrityl tetranitrate,    -   sulfisoxazole and erythrityl tetranitrate,    -   mitiglinide or levosimendan and erythrityl tetranitrate,    -   mitiglinide or levosimendan and zonisamide,    -   mitiglinide or levosimendan and terbinafine,    -   mitiglinide or levosimendan and risedronate,    -   mitiglinide or levosimendan and methyclothiazide,    -   methyclothiazide or zonisamide and sulfisoxazole,    -   terbinafine or risedronate and sulfisoxazole,    -   terbinafine or risedronate and mepacrine,    -   terbinafine or risedronate and acamprosate,    -   terbinafine or risedronate and rifabutin,    -   tadalafil or enprofylline or oxtriphylline and phenformin,    -   zonisamide or methyclothiazide and argatroban or cefinenoxime,    -   risedronate and argatroban or cefinenoxime,    -   zonisamide or methyclothiazide and cinnarizine or benidipine or        paramethadione or amlodipine,    -   acamprosate and cinnarizine or benidipine or paramethadione or        amlodipine,    -   zonisamide or methyclothiazide and ciclopirox,    -   sulfisoxazole and amobarbital,    -   zonisamide or methyclothiazide and amobarbital,    -   sulfisoxazole and cefotetan,    -   zonisamide or methyclothiazide and cefotetan,    -   zonisamide or methyclothiazide and erythrityl tetranitrate.

Specific examples of preferred compositions of the invention compriseone of the following drug combinations for combined, separate orsequential administration:

-   -   baclofen and aminocaproic acid,    -   baclofen and levosimendan,    -   aminocaproic acid and sulfisoxazole,    -   aminocaproic acid and terbinafine,    -   aminocaproic acid and levosimendan,    -   levosimendan and sulfisoxazole,    -   levosimendan and terbinafine,    -   eplerenone and levosimendan,    -   eplerenone and sulfisoxazole,    -   eplerenone and fenoldopam,    -   sulodexide and levosimendan,    -   sulodexide and sulfisoxazole,    -   sulodexide and sulfisoxazole,    -   torasemide and aminocaproic acid,    -   torasemide and levosimendan,    -   carbamazine and acamprosate,    -   sulodexide and fenoldopam, or eplerenone and sulodexide.

As illustrated in the experimental section, compositions comprising atleast aminocaproic acid or levosimendan provide substantial therapeuticand biological effect to improve Alzheimer's disease in human subjects.These compositions efficiently prevent the toxic effects of amyloid bprotein or peptide on human cells and represent novel and potent methodsfor treating such disorder.

In another preferred embodiment, the compositions according to theinvention comprise a combination of at least three compounds, or saltsor prodrugs or derivatives of any purity or sustained releaseformulations thereof, for simultaneous, separate or sequentialadministration for combinatorial treatment of Alzheimer's disease (AD)or a related disorder in a subject in need thereof.

Therapeutic approaches according to the invention may use drugs alone ordrug combinations, in conjunction with any other therapy targeting thesame pathway or having distinct modes of action.

In particular embodiments, the compositions of the present inventionmight further comprise a drug or drugs, which already exist or could bedeveloped, that bind to or modulate the activity of a protein encoded bya gene selected from ABAT, ABCA1, ABIl, ABL1, ACAT, ACC2, ACCN1,ADAMTS12, ADCY2, ADIPOQ, ADIPOR1/R2, ADORA1/2A/2B, ADRA1A/2, ADRB1/2,AGPAT5, AlP4, AKAP2, AKR1C2, AKT, ALDH2, ALOX12, ALOX5, ANG2, ANK1,ANKRA, ANXA1, APBA1, APBA2BP, APOA1, APOER2, ARHGAP17, ARHGAP26,ATG5/7/12, ATM, ATP10A, ATP1A1, ATP2A3, ATP2B1, ATP6V1C1, ATR, AUH,BACE1, BAD, BAI3, BASSOON, BAX, BCAR1, BCL2, BDNF, BECLIN1, BIN1, BKchannels (KCNMA1, KCNMB1), BMP3A, BRCA1, CA10, CACNA1C/2D3/2D4, CADPS2,CALM1-5 (calmodulin), CAMK1D, CAMKK2, CASK, CASR, CAST, CBL, CD36, CD44,CDC2, CDC42, CDC42BPB, CDC42EP3, CDH1/2/13, CDK5, CDKN1A, CHAT, CHK1,CHRM1-5, CHRNA1-7/9/10, CIT (citron), CK1, CNGB3, CNTFR, COL4A2, CPT,CRAM, CREB, CRMP, CSH1, CTNNA2, CTNNB1, CTTN (cortactin), CUBN, CULLIN1,CYP7B1, CYSLTR1/R2, DAB1, DCC, DEPDC2, DGKB/H/Z, DHCR7, DHFR, DLG2/4,DNAJB9, DOCK3, DRD2/5, DYN1/3, EDG1-8, EDN1/2, EDNRA/B, EFNA1/2/4/5/7(ephrin A), EFNB1/2/3 (ephrin B), EHHADH, ELAVL2, ENPP2 (autotaxin),ENPP6, EPHA3, EPHBR1/2/3/4/6, ERBB2/4, ERK1/2, ESRRG, ETFA, EZR, F2,F2R, FAS, FDPS, FES, FGF1/2, FKBP12/12.6, FLNA, FLT1 (VEGFR1), FLT4,FOXO1/3A, FRAP (MTOR), FTO, FYN, FZ2, GABBR1/2, GABRA2/G2, GADD45, GATT,GATA3, GH1, GIPC1/2, GLRA1, GLUD1, GNA12/13, GNPTAB, GPC5, GPHN(gephyrin), GRIA2/3, GRID1/2, GRIK1/2, GRIN2B/3A, GRIP1/2, GRK2/5,GRM3/5/6/7/8, GRP170, GSK3B, HAPLN1, HAS1-3, HCRTR2, HIF1A, HIPK2, HK2,HMOX1, HOMER1/2/3, HSD11B1, HSP90B1, HSPA5, HTR1A/1B/1D, HYAL1/2/3, IDE,IL20RA/B, IL6ST, IL8, IMPDH1/2, INS, INSR, IRF1, ITB1, ITGA1/6, ITGB1,ITPR1, JNK1, KALRN (kalirin), KCNA2/D2, KCNH2, KCNIP1/2, KCNJ11, KCNJ12,KCNJ3, KCNMA1, KCNMB1-4, KDR (VEGFR2), KTN1, KYNU, LAMA1, LDLR, LEP(LEPTIN), LEPR, LIFR, LIN7A/B/C (VELI1/2/3), L1PL2, LKB1, LRP1, LRP2(megalin), LTBP2, LYN, MAD1L1, MAML3, MAOA/B, MAT2B, MCC1, MDM1, MEl,MET, MGST2, MINT1, MLLT4 (afadin), MMP2, MMP9, MOESIN, MTR, MUC1,MUNC13/18A, MYO6, MYOL, NADPH oxidase, NAV1, NBEA, NCAM1, NCK1/2, NEDD9,NF2 (merlin), NFKB1, NFKBIB, NGEF (ephexin), NGF, NGFR, NHERF, NIL16,NLGN1, NOC2, NOS1/2A/3, NOTCH1/2/3, NPC1/2, NPIST, NR112, NR3C1, NR3C2,NRG1/3, NRP1/2, NRX3, NTF3/5, NTN1 (netrin 1), NTRK2 (TRKB), NWASP,OPCML, OPRK1, OPRM, OPRS1, OSBPL3/10, P2RY1, P2RY12, PAELR, PAIL/2,PAK1/6/7, PALLD, PAP1, PARK2, PC, PCAF, PCTP, PDE11A, PDE1A, PDE3A/3B,PDE4A/4B/4D, PDE5A, PDE6D, PDGFA/B, PDGFRA/B, PI3K, PIAS1, PICALM,PICK1, PIK3C3, PIP5K, PITPNC1, PKCA, PKCD, PLA1A/2, PLAT, PLAU, PLCB1,PLD1/2, PLEXA1, PLG, PLN, PLXDC2, PML, POP2, PPARA, PPARD, PPARG,PPARGC1B, PPFIBP1, PPP1CA, PPP3CA (calcineurin), PRDX5/6, PRKAA (AMPK),PRKACA, PRKG1, PRL, PTGER1, PTGFR, PTGS2, PTN, PTP1B, PTPN11, PTPRF,PTPRG, PTPRM, PVRL1, PXN (paxillin), PYK2, RAB3B, RAC1, RACK1, RAP1,RASGRF2, RBPJ, RDX (radixin), RELN, RGNEF, RHEB, RHOA, RHOG, RIM2,RIMS1/2, ROBO2, ROCK1/2, ROR2, RPH3A (rabphilin), RPH3AL, RPS6KA1, RPS6KB2, RTN1, RXR/RAR, RYR3, SACM1L, SAPAP, SAPK3, SCARB1, SCHIP1,SCN1A/1B, SCNN1D/1G, SEC24D, SEMA3A/3C/3E/4C, SGPP2, SH3BP5, SIAH1A,SIL1, SLC12A1/2/5, SLC1A2, SLC25A21, SLC6A1/A18, SLC8A1/A2/A3, SLC9A1,SLIT1, SLN, SMAD3/4, SNAP25, SNCA, SNCAIP, SORBS2, SORCS2, SPLA2,SPOCK1, SPP1 (osteopontin), SRC, SRD5A1, SREBF1/F2, SRGAP3, STAT3,STX1A/2 (syntaxins), STXBP6, SUM1, SV2C, SYN1, SYNJ1/2 (synaptojanin),SYT12, SYTL4 (granuphilin), TACE, TACR1, TBR1, TBXA2R, TGFBR1/R2/R3,THBS1/2, THEM2, THRA/B, TIAM1, TIMP2, TLL2, TOP2A, TP53, TP63, TR10,TRPC3/4/5, TSC1/2, TSPO, UBE2A, ULK4, UNC13C, UNC5C, VAMP2/5, VCL(VINCULIN), VDAC1, VEGFA/C, VEGFR1, VMAT, VPS15, WASPIP, WAVE, WNT1A/5A,WWOX, XANTHINE OXIDASE, YAP and YES1.

The sequences of all of the above listed genes and proteins areavailable from gene libraries and can be isolated by techniques known inthe art. The activity of these genes and proteins can also be assessedby techniques known in the art.

The invention also describes these supplementary drugs that can be usedto modulate target genes and proteins. We have identified particulardrugs which, either alone, or in combination(s), modulate the pathwaysdescribed above, and may be used to treat Alzheimer's disease or relateddisorders.

In a preferred embodiment, the compositions of the invention may furthercomprise at least one drug selected from an inhibitor ABAT, (preferably,vigabatrin), and/or an inhibitor ABL1 (preferably imatinib), and/or aninhibitor of ACAT (preferably, hesperetin), and/or a modulator of ADCY2(preferably, vidarabine), and/or a modulator of adenosine ADORA1/2A/3receptors (preferably selected from clofarabine and defibrotide), and/ora modulator of adrenergic ADRA receptors (preferably selected frompropericiazine, methotrimeprazine, mephentermine and dipivefrin), and/ora modulator of adrenergic ADRB receptors (preferably selected fromguanethidine, bethanidine, bitolterol and procaterol), and/or aninhibitor of ALOX5/12 (preferably selected from diethylcarbamazine andmasoprocol), and/or an inhibitor of ATP1A1 (preferably, deslanoside andomeprazole), and/or an activator of autophagy (preferably, trehalose),and/or an inhibitor of CA10 (preferably, methazolamide), and/or amodulator of calcification (preferably selected from foscarnet, galliumnitrate, calcifediol, calcitonin, calcitriol, clodronic acid,dihydrotachysterol, elcatonin, etidronic acid, ipriflavone andteriparatide acetate), and/or a modulator of CALM1 (calmodulin)(preferably, aprindine), and/or a modulator of CD44 (preferably selectedfrom eflornithine and benzbromarone), and/or a chemical chaperon(preferably selected from arabitol and mannitol), and/or a modulator ofmuscarinic CHRM receptors (preferably selected from cyclopentolate,oxyphencyclimine, trospium and isofluorophate), and/or an antagonist ofnicotinic acetylcholine CHRNA receptors, which is not able to crossblood-brain-barrier (preferably selected from pancuronium, pipecuronium,rapacuronium, rocuronium, succinylcholine, vecuronium, atracurium,cisatracurium, doxacurium, mecamylamine, metocurine, mivacurium andneomycin), and/or an inhibitor of CNGB3 (preferably, amiloride), and/ora modulator of CYSLTR1/2, PTGER1, PTGFR and TBXA2R eicosanoid receptors(preferably selected from travoprost, montelukast, cinalukast,amlexanox, carboprost tromethamine, bimatoprost and ridogrel), and/or aninhibitor of DHFR (preferably, pyrimethamine and triamterene), and/or amodulator of dopamine DRD2 receptor (preferably selected fromdihydroergotamine and cabergoline), and/or an agonist of dopaminereceptor DRD5 (preferably, fenoldopam), and/or an inhibitor of EDNRA(preferably selected from sulfamethoxazole and gentamicin), and/or amodulator of ENPP2 (autotaxin) (preferably, L-histidine), and/or aninhibitor of ERBB2 (preferably, lapatinib), and/or a modulator of F2thrombin (preferably selected from sulodexide, ximelagatran, warfarin,phenprocoumon, enoxaparin, ardeparin, fondaparinux, latamoxef,bacitracin, ticlopidine and erdosteine), and/or an inhibitor of FDPS(preferably, alendronate), and/or a modulator of GABRA2 (preferablyselected from phenobarbital, methohexital, cefotiam, clomethiazole,thiopental, lubiprostone and aztreonam), and/or an antagonist of GRIK1(preferably, topiramate), and/or a modulator of GSK3B activity(preferably selected from albuterol and metaraminol), and/or a modulatorof HIF1A signalling (preferably selected from meloxicam, topotecan,deferoxamine, usnic acid, hydralazine, deferiprone, dibenzoylmethane,avobenzone, dinoprostone, epoprostenol, 2-oxoglutarate and mimosine),and/or an inhibitor of HK2 (hexokinase II) (preferably selected fromquinine, gabexate, bifonazole and clotrimazole), and/or a modulator ofHMOX1 (preferably selected from auranofin, hematin/hemin and hemearginate), and/or a modulator of HTR1B/1D receptors (preferably selectedfrom ergotamine and eletriptan), and/or an inhibitor of IMPDH1 andIMPDH2 (preferably, thioguanine), and/or a modulator of integrins ITGA/B(preferably, rabeprazole), and/or an inhibitor of KCND2 potassiumchannel (preferably, lidocaine), and/or an inhibitor of KCNH2 potassiumchannel (preferably, ibutilide), and/or a modulator of KCNMA1(preferably selected from cromoglicate, ethinamate, ketoconazole,chlorzoxazone, unoprostone, hesperitin, bendroflumethiazide,benzthiazide, chlorothiazide, cyclothiazide, diazoxide,hydroflumethiazide, quinethazone and trichlormethiazide), and/or amodulator of MGST2 (preferably, balsalazide), and/or a modulator of MMP2and MMP9 (preferably, candoxatril), and/or a modulator of mitochondrialpermeability transition pore formation (preferably selected fromcarbenoxolone and ciprofloxacin), and/or an inhibitor of MTOR(preferably, rapamycin), and/or a modulator of NOS1/2A/3 (preferablyselected from propylthiouracil, thiethylperazine and ketotifen), and/ora modulator of NR3C1 receptor signalling (preferably selected frommetyrapone and mometasone), and/or a modulator of NR3C2 receptor(preferably selected from eplerenone and fludrocortisone), and/or aninhibitor of NRP2 (preferably, pegaptanib), and/or a modulator of OPCML(preferably, alfentanil), and/or a modulator of OPRK1 and OPRS1(preferably selected from buprenorphine and pentazocine), and/or OPRM(preferably, levallorphan), and/or a modulator of oxidativephosphorylation (preferably selected from almitrine, erythromycin,kanamycin and cerulenin), and/or an inhibitor of P2RY1 and/or P2RY12receptors (preferably, tirofiban), and/or an inhibitor of PDE11A, PDE4Aand PDE5A phosphodiesterases (preferably selected from mesembrine,milrinone and anagrelide), and/or an inhibitor of PDE3A/3B and PDE4A/4Bphosphodiesterases and an activator of BK channels (preferably,cilostazol), and/or a modulator of PDGFRA/B receptors (preferablyselected from becaplermin, streptomycin, delphinidin, cyanidin andfumagillin), and/or a modulator of PLA2 (preferably selected fromniflumic acid, hydrocortamate and netilmicin), and/or a modulator ofPLAT (preferably, sodium phenylbutyrate), and/or a modulator of PLD2(preferably, ambrisentan), and/or an inhibitor of PLG (preferably,aminocaproic acid), and/or a modulator of PPARD (preferably, icosapent),and/or a modulator of PPARG (preferably, phenylbutyrate), and/or amodulator of PRKG1 (preferably selected from nitroprusside,nitroglycerin and paricalcitol), and/or an inhibitor of PTP1B(preferably, tiludronate), and/or a modulator of RHOA/RAC (preferablyselected from chlorthalidone, hydrochlorothiazide, clomocycline,lymecycline, natamycin, amphotericin B, cefalexin, cephaloridine,cefuroxime, dicloxacillin), and/or a modulator of RXR/RAR (preferably,tazarotene), and/or an antagonist of SCN1A/B sodium channels(preferably, fosphenyloin), and/or an inhibitor of SLC12A1 (preferably,bumetanide), and/or an inhibitor of SLC6A1 (preferably, tiagabine),and/or a modulator of SLC9A1 (preferably, buclizine), and/or aninhibitor of SRD5A1 (preferably, dutasteride), and/or an antagonist ofTACR1 (preferably selected from aprepitant and vapreotide), and/or amodulator of TGFB signalling (preferably, aliskiren), and/or a modulatorof THRA/B (preferably selected from liothyronine), and/or an inhibitorof TOP2A (preferably, lucanthone), and/or a modulator of TSPO(preferably selected from flunitrazepam and temazepam), and/or amodulator of VDAC1 (preferably, dihydroxyaluminium), and/or an inhibitorof VEGFR1 (preferably, sunitinib), and/or a modulator of vitamin Kmetabolism (preferably selected cefinetazole, cefamandole andcefoperazone), and/or an inhibitor of VMAT (preferably selected fromtetrabenazine, deserpidine and nitisinone), and/or an inhibitor ofvoltage gated calcium channels (CACNA) (preferably selected fromlercanidipine, pregabalin, mibefradil, aranidipine, bamidipine,bencyclane, bepridil, clentiazem, efonidipine, elgodipine, etafenone,fendiline, flunarizine, gallopamil, isradipine, lacidipine, lidoflazine,lomerizine, manidipine, nicardipine, nilvadipine, nimodipine,nisoldipine, nitrendipine, perhexyline, prenylamine, semotiadil andterodiline), and/or an inhibitor of YES1, SRC and EPHA3 (preferably,dasatinib).

Other therapies used in conjunction with drug(s) or drug(s)combination(s) according to the present invention, may comprise one ormore drug(s) that ameliorate symptoms of Alzheimer's disease or drug(s)that could be used for palliative treatment of Alzheimer's disease.Preferably, said one or more drug(s) is/are selected from 3APS, AAB-001,ABT-089, ABT-126, AC-3933, ACC-001, Acetaminophen, AFFITOPE AD01,AFFITOPE AD02, alpha-lipoic acid, alpha-tocopherol, AN1792, anti-Abeta,AQWO51, Aripiprazole, Atomoxetine, Atorvastatin, AVE1625, AVP-923,AZD0328, AZD3480, Bapineuzumab, BAY94-9172 (ZK 6013443), Bifeprunox,Bioperine, BMS-708163, BRL-049653, Bryostatin, CAD106, Celecoxib,CERE-110, Cerebrolysin, CHF 5074, Choline, Circadin, Citalopram,Coenzyme Q, Copper, CTS21166, Curcumin, CX516 (Ampalex), CX717,Cyclophosphamate, DCB-AD1, Dextroamphetamine, DHA (DocosahexaenoicAcid), Digoxin, Dimebon (Latrepirdine), Divalproex, DMXB-A, Donepezil,Doxycycline, Egb 761, EHT 0202 tazolate, ELND005 (scyllo-inositol), EPAX1050TG, Ergoloid mesylate, Epigallocatechin-Gallate, Escitalopram,Estradiol, Estrogen, Etanercept, EVP-6124, EVT101, Exelon, Fish oil,FK962, florpiramine F 18, Folate+Vitamin B6+ Vitamin B21, Gabapentin,Galantamine, Gemfibrozil, Ginkgo biloba extracts (for example EGb 761 orCP401), improved extracts of Ginkgo biloba (for example enriched inactive ingredients or lessened in contaminant) or drug containing Ginkgobiloba extracts (for example Tanakan or Gingkor fort), Glucose,L-Glutamic Acid, GSI 136, GSI-953, GSK239512, GSK933776A, Haloperidol,HF0220, Huperzine A, hydrocodone/APAP, Ibuprofen, IFN-alpha2A,Indomethacin, Insulin, Intravenous Immunoglobulin, Ketasyn, Lecozotan,Leuprolide, Levodopa, Lipoic Acid, Lithium, Lorazepam, Lovostatin,Lutein, LY2062430 (solanezumab), LY2811376, LY450139, LY451395,MABT5102A, Malate, Masitinib (AB1010), Medroxyprogesterone, Melatonin,MEM 1003, MEM 3454, Memantine, Methylene blue, Methylphenidate,Mifepristone, MK0249, MK0677, MK0952, MK0952, MK3328, Modafinil,MPC-7869, NADH, Naproxen, Nefiracetam, Neptune Krill Oil, Neramexane,NIC5-15, Nicoderm Patch, Nicotinamide (vitamin B3), Novasoy, NPO31112,NS 2330, NSA-789, NSAIDs, Olanzapine, omega-3 polyunsaturated fattyacids (EPA+DHA), ONO-2506P0, Oxybate, Panax Ginseng, PAZ-417, PBT2,Perphenazine, PF-04360365, PF-04447943, PF-04494700, Phenserine,Phosphatidylserine, Pitavastatin, Posiphen, PPI-1019 (APAN),Pravastatin, Prazosin, Prednisone, Progesterone, PRX-03140, PYM50028,Quetiapine, R1450, Raloxifene, Ramipril, Rasagiline, Razadyne,resveratrol, rifampicin, risperidone, Rivastigmine, RN1219, R05313534,Rofecoxib, Rosiglitazone, Salvia officinalis (sage), SAM-315, SAM-531,SAM-760, SB-742457, Selenium, Sertraline, SGS-742, Simvastatin,SK-PC-B70M, Solanezumab, SR57667B, SRA-333, SRA-444, SSR180711c, ST101,T-817MA, Tacrine, Tarenflurbil, Testosterone, Tramiprosate (3APS),Trazodone, TRx0014 (methylthioninium chloride), Tryptophan, V950,Valproate, Varenicline, Vitamin C, Vitamin E, VP4896, Xaliproden,Zeaxanthin, Zolpidem, and ZT-1 (DEBIO-9902 SR).

The invention also relates to a method of treating Alzheimer's diseaseor a related disorder, the method comprising simultaneously, separatelyor sequentially administering to a subject in need thereof a drugcombination as disclosed above.

A further object of this invention is a method of treating Alzheimer'sdisease or a related disorder, the method comprising simultaneously,separately or sequentially administering to a subject in need thereof adrug combination that modulates synapse function and/or a drug thatmodulates angiogenesis and/or a drug that modulates cell stressresponse.

A further object of the invention resides in a method of selecting adrug for combinatorial treating Alzheimer's disease or a relateddisorder, the method comprising a step of testing a candidate drug foractivity on synapse function and/or angiogenesis and/or cellular stressresponse and selecting a candidate drug that ameliorates synapsefunction, attenuates angiogenic dysregulation and modulates cellularstress response.

In another embodiment, the invention relates to a method of selecting acomposition for treating Alzheimer's disease or a related disorder, themethod comprising preparing a combination of a drug that modulatessynapse function and/or a drug that attenuates angiogenic dysregulationand/or a drug that modulates cell stress response, for simultaneous,separate or sequential administration to a subject in need thereof.

In another preferred embodiment, the invention relates to a method oftreating Alzheimer's disease or a related disorder, the methodcomprising simultaneously, separately or sequentially administering to asubject in need thereof a drug that modulates synapse function and/or adrug that modulates angiogenesis and/or a drug that modulates cellstress response.

The composition of the invention may be administered repeatedly to thesubject.

The compositions of the invention typically comprise one or severalpharmaceutically acceptable carriers or excipients. The duration of thetherapy depends on the stage of the disease being treated, thecombination used, the age and condition of the patient, and how thepatient responds to the treatment. The dosage, frequency and mode ofadministration of each component of the combination can be controlledindependently. For example, one drug may be administered orally whilethe second drug may be administered intramuscularly. Combination therapymay be given in on-and-off cycles that include rest periods so that thepatient's body has a chance to recover from any as yet unforeseenside-effects. The drugs may also be formulated together such that oneadministration delivers all drugs.

The administration of each drug of the combination may be by anysuitable means that results in a concentration of the drug that,combined with the other component, is able to correct the functioning ofpathways implicated in AD.

While it is possible for the active ingredients of the combination to beadministered as the pure chemical it is preferable to present them as apharmaceutical composition, also referred to in this context aspharmaceutical formulation. Possible compositions include those suitablefor oral, rectal, topical (including transdermal, buccal andsublingual), or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration.

More commonly these pharmaceutical formulations are prescribed to thepatient in “patient packs” containing a number dosing units or othermeans for administration of metered unit doses for use during a distincttreatment period in a single package, usually a blister pack. Patientpacks have an advantage over traditional prescriptions, where apharmacist divides a patient's supply of a pharmaceutical from a bulksupply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in traditionalprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions. Thus, theinvention further includes a pharmaceutical formulation, as hereinbefore described, in combination with packaging material suitable forsaid formulations. In such a patient pack the intended use of aformulation for the combination treatment can be inferred byinstructions, facilities, provisions, adaptations and/or other means tohelp using the formulation most suitably for the treatment. Suchmeasures make a patient pack specifically suitable for and adapted foruse for treatment with the combination of the present invention.

The drug may be contained in any appropriate amount in any suitablecarrier substance, and is may be present in an amount of 1-99% by weightof the total weight of the composition. The composition may be providedin a dosage form that is suitable for the oral, parenteral (e.g.,intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal,inhalant, skin (patch), or ocular administration route. Thus, thecomposition may be in the form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, osmoticdelivery devices, suppositories, enemas, injectables, implants, sprays,or aerosols.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

The controlled release formulations include (i) formulations that createa substantially constant concentration of the drug within the body overan extended period of time; (ii) formulations that after a predeterminedlag time create a substantially constant concentration of the drugwithin the body over an extended period of time; (iii) formulations thatsustain drug action during a predetermined time period by maintaining arelatively, constant, effective drug level in the body with concomitantminimization of undesirable side effects associated with fluctuations inthe plasma level of the active drug substance; (iv) formulations thatlocalize drug action by, e.g., spatial placement of a controlled releasecomposition adjacent to or in the diseased tissue or organ; and (v)formulations that target drug action by using carriers or chemicalderivatives to deliver the drug to a particular target cell type.

Administration of drugs in the form of a controlled release formulationis especially preferred in cases in which the drug, either alone or incombination, has (i) a narrow therapeutic index (i.e., the differencebetween the plasma concentration leading to harmful side effects ortoxic reactions and the plasma concentration leading to a therapeuticeffect is small; in general, the therapeutic index, TI, is defined asthe ratio of median lethal dose (LD50) to median effective dose (ED50));(ii) a narrow absorption window in the gastro-intestinal tract; or (iii)a very short biological half-life so that frequent dosing during a dayis required in order to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the drug in question. Controlled release may be obtainedby appropriate selection of various formulation parameters andingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the drug is formulated with appropriateexcipients into a pharmaceutical composition that, upon administration,releases the drug in a controlled manner (single or multiple unit tabletor capsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, microcrystalline cellulose, starches includingpotato starch, calcium carbonate, sodium chloride, calcium phosphate,calcium sulfate, or sodium phosphate); granulating and disintegratingagents (e.g., cellulose derivatives including microcrystallinecellulose, starches including potato starch, croscarmellose sodium,alginates, or alginic acid); binding agents (e.g., acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., stearic acid, silicas, or talc).Other pharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). A time delay material suchas, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology.

Several drugs may be mixed together in the tablet, or may bepartitioned. For example, the first drug is contained on the inside ofthe tablet, and the second drug is on the outside, such that asubstantial portion of the second drug is released prior to the releaseof the first drug.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, microcrystalline cellulose,calcium carbonate, calcium phosphate or kaolin), or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, liquid paraffin, or olive oil. Powders andgranulates may be prepared using the ingredients mentioned above undertablets and capsules in a conventional manner.

Controlled release compositions for oral use may, e.g., be constructedto release the active drug by controlling the dissolution and/or thediffusion of the active drug substance.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of drugs, or by incorporating the drug into an appropriatematrix. A controlled release coating may include one or more of thecoating substances mentioned above and/or, e.g., shellac, beeswax,glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more of the drugs ofthe claimed combinations may also be in the form of a buoyant tablet orcapsule (i.e., a tablet or capsule that, upon oral administration,floats on top of the gastric content for a certain period of time). Abuoyant tablet formulation of the drug(s) can be prepared by granulatinga mixture of the drug(s) with excipients and 20-75% w/w ofhydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of anaqueous suspension by addition of water are convenient dosage forms fororal administration. Formulation as a suspension provides the activeingredient in a mixture with a dispersing or wetting agent, suspendingagent, and one or more preservatives. Suitable suspending agents are,for example, sodium carboxymethylcellulose, methylcellulose, sodiumalginate, and the like.

Parenteral Compositions

The pharmaceutical composition may also be administered parenterally byinjection, infusion or implantation (intravenous, intramuscular,subcutaneous, or the like) in dosage forms, formulations, or viasuitable delivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants. The formulation andpreparation of such compositions are well known to those skilled in theart of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active drug(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active drug(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. The composition may include suspending, solubilizing,stabilizing, pH-adjusting agents, and/or dispersing agents.

The pharmaceutical compositions according to the invention may be in theform suitable for sterile injection. To prepare such a composition, thesuitable active drug(s) are dissolved or suspended in a parenterallyacceptable liquid vehicle. Among acceptable vehicles and solvents thatmay be employed are water, water adjusted to a suitable pH by additionof an appropriate amount of hydrochloric acid, sodium hydroxide or asuitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodiumchloride solution. The aqueous formulation may also contain one or morepreservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). Incases where one of the drugs is only sparingly or slightly soluble inwater, a dissolution enhancing or solubilizing agent can be added, orthe solvent may include 10-60% w/w of propylene glycol or the like.

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the activedrug(s) may be incorporated in biocompatible carriers, liposomes,nanoparticles, implants, or infusion devices. Materials for use in thepreparation of microspheres and/or microcapsules are, e.g.,biodegradable/bioerodible polymers such as polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine). Biocompatible carriersthat may be used when formulating a controlled release parenteralformulation are carbohydrates (e.g., dextrans), proteins (e.g.,albumin), lipoproteins, or antibodies. Materials for use in implants canbe non-biodegradable (e.g., polydimethyl siloxane) or biodegradable(e.g., poly(caprolactone), poly(glycolic acid) or poly(ortho esters)).

Rectal Compositions

For rectal application, suitable dosage forms for a composition includesuppositories (emulsion or suspension type), and rectal gelatin capsules(solutions or suspensions). In a typical suppository formulation, theactive drug(s) are combined with an appropriate pharmaceuticallyacceptable suppository base such as cocoa butter, esterified fattyacids, glycerinated gelatin, and various water-soluble or dispersiblebases like polyethylene glycols. Various additives, enhancers, orsurfactants may be incorporated.

Percutaneous and Topical Compositions

The pharmaceutical compositions may also be administered topically onthe skin for percutaneous absorption in dosage forms or formulationscontaining conventionally non-toxic pharmaceutical acceptable carriersand excipients including microspheres and liposomes. The formulationsinclude creams, ointments, lotions, liniments, gels, hydrogels,solutions, suspensions, sticks, sprays, pastes, plasters, and otherkinds of transdermal drug delivery systems. The pharmaceuticallyacceptable carriers or excipients may include emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

The emulsifying agents may be naturally occurring gums (e.g., gum acaciaor gum tragacanth).

The preservatives, humectants, penetration enhancers may be parabens,such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride,glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topicaladministration on the skin may also be used in connection with topicaladministration onto or close to the part of the body that is to betreated. The compositions may be adapted for direct application or forapplication by means of special drug delivery devices such as dressingsor alternatively plasters, pads, sponges, strips, or other forms ofsuitable flexible material.

Dosages and Duration of the Treatment

It will be appreciated that the drugs of the combination may beadministered concomitantly, either in the same or differentpharmaceutical formulation or sequentially. If there is sequentialadministration, the delay in administering the second (or additional)active ingredient should not be such as to lose the benefit of theefficacious effect of the combination of the active ingredients. Aminimum requirement for a combination according to this description isthat the combination should be intended for combined use with thebenefit of the efficacious effect of the combination of the activeingredients. The intended use of a combination can be inferred byfacilities, provisions, adaptations and/or other means to help using thecombination according to the invention.

Although the active drugs of the present invention may be administeredin divided doses, for example two or three times daily, a single dailydose of each drug in the combination is preferred, with a single dailydose of all drugs in a single pharmaceutical composition (unit dosageform) being most preferred.

The term “unit dosage form” refers to physically discrete units (such ascapsules, tablets, or loaded syringe cylinders) suitable as unitarydosages for human subjects, each unit containing a predeterminedquantity of active material or materials calculated to produce thedesired therapeutic effect, in association with the requiredpharmaceutical carrier.

Administration can be one to several times daily for several days toseveral years, and may even be for the life of the patient. Chronic orat least periodically repeated long-term administration will beindicated in most cases.

Additionally, pharmacogenomic (the effect of genotype on thepharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic)information about a particular patient may affect the dosage used.

Except when responding to especially impairing AD disease cases whenhigher dosages may be required, the preferred dosage of each drug in thecombination usually lies within the range of doses not above thoseusually prescribed for long-term maintenance treatment or proven to besafe in phase 3 clinical studies.

One remarkable advantage of the invention is that each compound may beused at low doses in a combination therapy, while producing, incombination, a substantial clinical benefit to the patient. Thecombination therapy may indeed be effective at doses where the compoundshave individually no substantial effect. Accordingly, a particularadvantage of the invention lies in the ability to use sub-optimal dosesof each compound, i.e., doses which are lower than therapeutic dosesusually prescribed, preferably ½ of therapeutic doses, more preferably⅓, ¼, ⅕, or even more preferably 1/10 to 1/100 of therapeutic doses. Atsuch sub-optimal dosages, the compounds alone would be substantiallyinactive, while the combination(s) according to the invention are fullyeffective.

A preferred dosage corresponds to amounts from 1% up to 50% of thoseusually prescribed for long-term maintenance treatment. The mostpreferred dosage may correspond to amounts from 1% up to 10% of thoseusually prescribed for long-term maintenance treatment.

Specific examples of dosages of drugs for use in the invention areprovided below:

-   -   Aminocaproic acid from about 0.05 to 15 g per day,    -   Levosimendan from 0.05 to 4 mg per day,    -   amlodipine orally from about 0.05 to 1 mg per day,    -   clopidogrel orally from about 0.75 to 7.5 mg per day,    -   tadalafil orally from about 0.05 to 0.5 mg per day,    -   cilostazol orally from about 1 to 10 mg per day,    -   terbinafine orally from about 2.5 to 25 mg once or twice daily,    -   leflunomide orally from about 0.25 to 2.5 mg per day,    -   cinacalcet orally from about 0.3 to 3 mg per day,    -   acamprosate orally from about 7 to 70 mg three times daily,    -   methimazole orally from about 0.05 to 1.5 mg per day,    -   mepacrine orally from about 3 to 30 mg per day,    -   phenformin orally from about 0.5 to 5 mg per day,    -   baclofen orally from about 0.4 to 8 mg per day administered in        two or three divided doses,    -   rifabutin orally from about 6 to 60 mg per day,    -   amobarbital orally from about 0.06 to 15 mg per day,    -   cefotetan orally from about 0.01 to 0.4 mg per day,    -   dyphylline orally from about 6 to 60 mg per day in two or three        divided doses,    -   methyclothiazide orally from about 0.025 to 1 mg per day,    -   risedronate orally from about 0.05 to 3 mg per day,    -   etomidate orally from about 0.6 to 6 mg per day,    -   torasemide orally from about 0.05 to 4 mg per day,    -   zonisamide orally from about 1 to 40 mg per day.

It will be understood that the amount of the drug actually administeredwill be determined by a physician, in the light of the relevantcircumstances including the condition or conditions to be treated, theexact composition to be administered, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thechosen route of administration. Therefore, the above dosage ranges areintended to provide general guidance and support for the teachingsherein, but are not intended to limit the scope of the invention.

The following examples are given for purposes of illustration and not byway of limitation.

EXAMPLES

I. The Compounds and Combinations Thereof Prevent Toxicity of Aβ₂₅₋₃₅Peptide

In this first series of experiments, candidate compounds have beentested for their ability to prevent or reduce the toxic effects ofAβ₂₅₋₃₅ peptide. The drugs are first tested individually, followed byassays of their combinatorial action. The effect is determined onvarious cell types, to further illustrate the activity of the compounds.

In AD, the APP protein forms aggregates of insoluble 0-pleated sheets offibrillar Abeta protein (amyloid). The conformational change fromsoluble to fibrillar forms seems to be a spontaneous event that isincreased with higher concentrations of Abeta, so any production oflarger amounts of Abeta than normal (or production of the larger, lesssoluble forms of Abeta) will tend to increase plaque formation. Once theAbeta plaque has started to form, other molecules can interact with thenascent plaque to produce eventually the mature plaque with itsassociated areas of neuronal cell death. Considering this, we have givenpriority to testing the effects of the drugs on the viability of thecells exposed to the amyloid βprotein.

I.1 Protection Against the Toxicity of Aβ₂₅₋₃₅ Peptide on CorticalNeurons Cell Culture

Primary rat cortical neurons are cultured as described by Singer et al.,1999. Briefly pregnant female rats of 15 days gestation are killed bycervical dislocation (Rats Wistar; Janvier) and the foetuses removedfrom the uterus. The cortex are removed and placed in ice-cold medium ofLeibovitz (L15; Invitrogen) containing 1% of Penicillin-Streptomycin(PS; Invitrogen) and 1% of bovine serum albumin (BSA; Sigma). Cortex aredissociated by trypsinisation for 20 min at 37° C. (Trypsin EDTA 1X;Invitrogen) diluted in PBS without calcium and magnesium. The reactionis stopped by the addition of Dulbecco's modified Eagle's medium (DMEM;Invitrogen) containing DNAase I grade II (0.1 mg/ml; Roche Diagnostic)and 10% of foetal calf serum (FCS; Invitrogen). Cells are thenmechanically dissociated by 3 passages through a 10 ml pipette. Cellsare then centrifuged at 180×g for 10 min at 10° C. The supernatant isdiscarded and the cells of pellet are re-suspended in a defined culturemedium consisting of Neurobasal (Invitrogen) supplemented with B27 (2%;Invitrogen), L-glutamine (0.2 mM; Invitrogen), 1% of PS solution and 10ng/ml of Brain-derived neurotrophic factor (BDNF, Pan Biotech). Viablecells are counted in a Neubauer cytometer using the trypan blueexclusion test. Cells are seeded at a density of 30 000 cells/well in 96well-plates (wells are pre-coated with poly-L-lysine (10 μg/ml; Sigma))and are cultured at 37° C. in a humidified air (95%)/CO2 (5%)atmosphere.

After 6 days of culture, cells are incubated with drugs (5concentrations). After 1 hour, cells are intoxicated by 20 μM ofbeta-amyloid (25-35; Sigma) in defined medium without BDNF but togetherwith drugs. Cortical neurons are intoxicated for 2 days. Two independentcultures are performed per condition, 6 wells per condition.

Neurites Length Quantification

Cells are fixed with a cool solution of ethanol (95%) and acetic acid(5%) for 10 min. After permeabilization with 0.1% of saponin, cells areblocked for 2 h with PBS containing 10% goat serum. Cells are thenincubated with monoclonal antibody directed against the microtubuleassociated protein 2 (MAP-2; Sigma). This antibody reveals specificallycell bodies and neurites. The secondary antibody used is an Alexa Fluor488 goat anti-mouse IgG (Molecular probe). Nuclei of neurons arerevealed by a fluorescent dye (Hoechst solution, SIGMA). Twenty picturesare taken per well, using InCell Analyzer™ 1000 (GE Healthcare) atmagnification 20×. All images are taken in the same conditions. Neuriteslength is quantified using Developer software (GE Healthcare).

Results

Results presented in FIG. 1 are extracted from two independent cultures,6 wells per condition. All values are expressed as mean±s.e.mean. Abilateral Student's t test analysis is performed on raw data. Resultsare expressed in percentage of neurites length, compared to the control(vehicle).

Drugs were incubated with rat primary cortical neurons one hour beforeAbeta₂₅₋₃₅ 20 μM intoxication that lasts 2 days (36).

Two days after this incubation the network of neurites length wasquantified, reflecting axonal cell growth. The results show that thetested drugs clearly exert a neuroprotective effect against Abeta₂₅₋₃₅intoxication (FIG. 1 and FIG. 2).

I.2. Protection Against the Toxicity of Aβ₂₅₋₃₅ Peptide on EndothelialCerebral Cells Cell Culture

Primo culture of rat endothelial cerebral cells (Vect-Horus SAS,Marseille) is cultivated on passage 0. At confluence, endothelial cellsare dissociated with trypsin EDTA (Pan Biotech Ref: P10-023100). Cellsare seeded at a density of 25 000 cells/well in 96 well-plates (wellsare coated with 30 g1 of type I rat collagen at 1.5 mg/ml, Vect-HorusSAS, Marseille) and are cultured in MCBD 131 medium (M-131-500,Invitrogen) supplemented with 1% of microvascular growth supplement(MVGS, S-005-25, Invitrogen). Cells are cultured at 37° C. in ahumidified air (95%)/CO2 (5%) atmosphere. Half of the medium is changedevery other day with fresh medium.

After 4 days, drugs are added to the cell culture medium, at differentconcentrations, solved in DMSO 0.1% or water. A 1 hour pre-incubation isperformed, in a culture medium containing Dulbecco's modified Eagle'smedium (DMEM, Pan Biotech Ref: P04-03600), supplemented with 2% of fetalbovine serum (FBS; Invitrogen ref: 16000-036), 1% of L-glutamine (PanBiotech ref: P04-80100), 1% of Penicillin-Streptomycin (PS; Pan Biotechref: P06-07100), 0.1 mg/ml of Heparin (Sigma), 10 ng/ml of epidermalgrowth factor (EGF, Invitrogen) and 10 ng/ml of vascular endothelialgrowth factor (VEGF, PHG0146, Invitrogen).

Cells are then intoxicated with 30 μM of β-amyloid (25-35; Sigma)together with drugs in the same culture medium. Cells are thenintoxicated during 3 days.

Lactate Dehydrogenase (LDH) Activity Assay

For each culture, after 3 days of intoxication, the supernatant iscollected and analyzed with Cytotoxicity Detection Kit (LDH, RocheApplied Sciences). This colorimetric assay for the quantification ofcell death is based on the measurement of lactate dehydrogenase (LDH)activity released from the cytosol of damaged cells into thesupernatant. The optic density (DO) is assessed by spectrophotometer at492 nm wavelength by a multiscan apparatus (Thermo, Ref Ascent).

Results

Results presented in FIG. 3 are extracted from two independent cultures,6 wells per condition. All values are expressed as mean±s.e.m. Abilateral Student's t test analysis is performed on raw data. Resultsare expressed in percentage of cell viability, compared to the control(vehicle).

Drugs are incubated with rat primary cerebral endothelial cells one hourbefore Aβ₂₅₋₃₅ 30 μM intoxication that lasts 3 days.

Three days after this incubation, LDH release in the culture medium isquantified, reflecting the level of cell death.

The results presented clearly show that the tested compounds exert apotent protective effect against this Aβ₂₅₋₃₅ intoxication (FIG. 3).

I.3. Protection Against the Toxicity of Aβ₂₅₋₃₅ Peptide onPheochromocytoma Cells PC12 Cell Culture

PC12 (Pheochromocytoma Rat, ATCC ref: CRL-1721) cells from ATCC (ATCCCRL-1721) were rapidly thawed in 37° C. water. The supernatant wasimmediately put in 9 ml of a PC12 proliferation medium containingDulbecco's modified Eagle's medium DMEM-F12 (Pan Biotech ref: P04-41450)with 15% heat-inactivated horse serum (Invitrogen ref: 16050-130), 2.5%of fetal bovine serum (FBS; Invitrogen ref: 16000-036), 1% of Penicillin10.000 U/ml and Streptomycin 10 mg/ml (PS; Pan Biotech ref: P06-07100)and 1% de L-glutamine 200 mM (Pan Biotech ref: P04-80100).

Cells were centrifuged (800 rounds/min, 4° C. for 5 min) and added in 5ml PC12 proliferation medium, viable cells were counted with a Malassezcell using the neutral red exclusion test (Sigma).

Then the cells were seeded at 3.10⁴ cells per cm² in PC12 proliferationmedium in 75 cm² plastic flasks (Greiner Ref: 658175) precoated withpoly-L-lysine (10 μg/ml, Sigma Ref: P2636).

Medium was changed every other day. After 3 days of culture, when cellsreached 80% of confluence, they were washed in HBSS without calcium andmagnesium (Pan Biotech Ref: P06-33500) and incubated in trypsin EDTA,(0.05%, Pan Biotech Ref: P10-023100). The enzymatic reaction was stoppedwith PC12 proliferation medium added by 0.5 mg/ml of DNAse 1 grade 2(Pan Biotech Ref: T60-37780100). Then, PC12 were centrifuged (800rounds/min at 4° C. for 10 min) and cells were seeded at the density of2.9 10⁴ per cm² in 175 cm² culture flask (Greiner Ref: 661195)pre-coated with poly-L-lysine.

Intoxication and MTT Viability Assay

PC12 cells (passage #2) are seeded on the basis of 3300 cells per cm² in96 well-plates (Greiner Ref: 655 180) pre-coated with poly-L-lysine(Sigma) Neurobasal medium (Invitrogen, Ref: 21103049) containing B27(2%, Invitrogen, Ref: 21103049), penicillin (50 U/ml)-streptomycin (50μg/ml) and glutamine (1%) and 50 ng/ml of NGF (Sigma Ref: N1408). NGFallows PC12 to differentiate in sympatic neuron-like cells.

After 5 days of culture, the medium is changed with neurobasal added byNGF (50 ng/ml), B27 without antioxidant, glutamine and antibiotics.After 24 h, cells are incubated for 1 hour with drugs at 5concentrations, 6 wells per conditions. After 1 hour of pre-incubation,cells are intoxicated by 10 μM of beta-amyloïd (25-35; Sigma) togetherwith drugs in the cell culture medium. 24 h later, cells are washed oncewith PBS (Pan Biotech, Ref: P04-36100) and the PC12 cell survival wasevaluated by MTT (3,[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide) viability test.

Cortical Neurons Cell Culture

Primary rat cortical neurons are cultured as described by Singer et al.,1999. Briefly pregnant female rats of 15 days gestation are killed bycervical dislocation (Rats Wistar; Janvier) and the foetuses removedfrom the uterus. The cortex are removed and placed in ice-cold medium ofLeibovitz (L15; Invitrogen) containing 1% of Penicillin-Streptomycin(PS; Invitrogen) and 1% of bovine serum albumin (BSA; Sigma). Cortex aredissociated by trypsinisation for 20 min at 37° C. (Trypsin EDTA 1X;Invitrogen) diluted in PBS without calcium and magnesium. The reactionis stopped by the addition of Dulbecco's modified Eagle's medium (DMEM;Invitrogen) containing DNAase I grade II (0.1 mg/ml; Roche Diagnostic)and 10% of foetal calf serum (FCS; Invitrogen). Cells are thenmechanically dissociated by 3 passages through a 10 ml pipette. Cellsare then centrifuged at 180×g for 10 min at 10° C. The supernatant isdiscarded and the cells of pellet are re-suspended in a defined culturemedium consisting of Neurobasal (Invitrogen) supplemented with B27 (2%;Invitrogen), L-glutamine (0.2 mM; Invitrogen), 1% of PS solution and 10ng/ml of Brain-derived neurotrophic factor (BDNF, Pan Biotech). Viablecells are counted in a Neubauer cytometer using the trypan blueexclusion test. Cells are seeded at a density of 30 000 cells/well in 96well-plates (wells are pre-coated with poly-L-lysine (10 μg/ml; Sigma))and are cultured at 37° C. in a humidified air (95%)/CO2 (5%)atmosphere.

After 6 days of culture, cells are incubated with drugs (5concentrations). After 1 hour, cells are intoxicated by 20 μM ofβ-amyloïd (25-35; Sigma) in defined medium without BDNF but togetherwith drugs. Cortical neurons are intoxicated for 2 days.

Lactate Dehydrogenase (LDH) Activity Assay

After 2 days of culture, the supernatant is collected and analyzed withCytotoxicity Detection Kit (LDH, Roche Applied Sciences). Thiscolorimetric assay for the quantification of cell death is based on themeasurement of lactate dehydrogenase (LDH) activity released from thecytosol of damaged cells into the supernatant. The optic density (DO) isassessed by spectrophotometer at 492 nm wavelength by a multiscanapparatus (Thermo, Ref Ascent). Results are expressed in percentage ofcell viability, compared to the negative control (vehicle).

Results

Results presented in FIGS. 4 and 5 are extracted from two independentcultures, 6 wells per condition. All values are expressed asmean±s.e.mean. A bilateral Student's t test analysis is performed on rawdata. Results are expressed in percentage of neurites length, comparedto the control (vehicle).

NGF-differentiated PC12 cells are incubated with drugs one hour beforeAbeta₂₅₋₃₅ 10 μM intoxication that lasts 24 hours.

One day after this incubation, the viability of NGF-differentiated PC12is quantified, using MTT assay. The results clearly show that prilocalnand amlodipin exert a strong neuroprotective effect against thisAbeta₂₅₋₃₅ intoxication (FIG. 4).

Rat primary cortical neurons were also incubated with compounds of theinvention one hour before Aβ₂₅₋₃₅ 20 μM intoxication that lasts 2 days.Two days after this incubation, LDH release in the culture medium isquantified, reflecting the level of cell death. The results presenteddemonstrate that compounds for use in the present invention exert asubstantial protective effect against this Aβ₂₅₋₃₅ intoxication (FIG.5).

I.4. Activity of Drug Combinations

In vitro assays are also carried out with several combinations of drugsmodulating synapse function and/or angiogenesis and/or cell stressresponse. Drugs are incubated in the same experimental conditions asdescribed above (see sections 1.1-1.3). The most efficient drugcombinations acting on the targets are summarized in Table 2.

TABLE 2 Neuro protective effect against the Drug combination Aβ₂₅₋₃₅intoxication phenformin and zonisamide + phenformin andmethyclothiazide + phenformin and acamprosate + phenformin andsulfisoxazole + baclofen and terbinafine + baclofen and risedronate +baclofen and sulfisoxazole + baclofen and zonisamide + baclofen andmethyclothiazide + baclofen and leflunomide + zonisamide anddyphylline + methyclothiazide and dyphylline + zonisamide andprilocaine + methyclothiazide and prilocaine + zonisamide andsulfisoxazole + terbinafine and sulfisoxazole + terbinafine andmepacrine + acamprosate and terbinafine + terbinafine and rifabutin +phenformin and tadalafil + zonisamide and argatroban + phenformin andclopidogrel + acamprosate and cinacalcet + sulfisoxazole andcinacalcet + terbinafine and argatroban + terbinafine and cefmenoxime +baclofen and clopidogrel + terbinafine and clopidogrel + risedronate andclopidogrel + zonisamide and cinnarizine + acamprosate and cinnarizine +zonisamide and ciclopirox + acamprosate and ciclopirox + sulfisoxazoleand amobarbital + zonisamide and amobarbital + sulfisoxazole andcefotetan + zonisamide and cefotetan + acamprosate and erythrityltetranitrate + zonisamide and erythrityl tetranitrate + sulfisoxazoleand erythrityl tetranitrate + mitiglinide and erythrityl tetranitrate +levosimendan and erythrityl tetranitrate + mitiglinide and zonisamide +levosimendan and zonisamide + mitiglinide and terbinafine + levosimendanand terbinafine + mitiglinide and risedronate + levosimendan andrisedronate + mitiglinide and methyclothiazide + levosimendan andmethyclothiazide + methyclothiazide and sulfisoxazole + zonisamide andsulfisoxazole + risedronate and sulfisoxazole + risedronate andmepacrine + risedronate and acamprosate + risedronate and rifabutin +enprofylline and phenformin + oxtriphylline and phenformin + zonisamideand cefmenoxime + methyclothiazide and argatroban + methyclothiazide andcefmenoxime + risedronate and argatroban + risedronate and cefmenoxime +zonisamide and cinnarizine + zonisamide and benidipine + zonisamide andparamethadione + zonisamide and amlodipine + methyclothiazide andcinnarizine + methyclothiazide and benidipine + methyclothiazide andparamethadione + methyclothiazide and amlodipine + acamprosate andbenidipine + acamprosate and paramethadione + acamprosate andamlodipine + methyclothiazide and ciclopirox + methyclothiazide andamobarbital + methyclothiazide and cefotetan + methyclothiazide anderythrityl tetranitrate + + indicates positive neuroprotective effectagainst the Aβ₂₅₋₃₅ intoxicationII. The Compounds Prevent Toxicity of Human Aβ₁₋₄₂

In this further series of experiments, candidate compounds have beentested for their ability to prevent or reduce the toxic effects of humanAβ₁₋₄₂. Aβ₁₋₄₂ is the full length peptide that constitutes aggregatesfound in biopsies from human patients afflicted with AD. The drugs arefirst tested individually, followed by assays of their combinatorialaction. The effect is determined on various cell types, to furtherdocument the activity of the compounds.

II.1. Protection Against the Toxicity of Aβ₁₋₄₂ on Human BrainMicrovascular Endothelial Cell Model

Human brain microvascular endothelial cell cultures were used to studythe protection afforded by candidate compounds on Aβ₁₋₄₂ toxicity. Humanbrain microvascular endothelial cerebral cells (HBMEC, ScienCell Ref:1000, frozen at passage 10) were rapidly thawed in a water bath at +37°C. The supernatant was immediately put in 9 ml Dulbecco's modifiedEagle's medium (DMEM; Pan Biotech ref: P04-03600) containing 10% offoetal calf serum (FCS; GIBCO ref 10270-106). Cell suspension wascentrifuged at 180×g for 10 min at +4° C. and the pellets were suspendedin CSC serum-free medium (CSC serum free, Cell System, Ref:SF-4Z0-500-R, Batch 51407-4) with 1.6% of Serum free RocketFuel (CellSystem, Ref: SF-4Z0-500-R, Batch 54102), 2% of Penicillin 10.000 U/mland Streptomycin 10mg/ml (PS ; Pan Biotech ref: P06-07100 batch133080808) and were seeded at the density of 20 000cells per well in 96well-plates (matrigel layer biocoat angiogenesis system, BD, Ref 354150,Batch A8662) in a final volume of 100 μl. On matrigel support,endothelial cerebral cells spontaneously started the process ofcapillary network morphogenesis (47). Three separate cultures wereperformed per condition, 6 wells per condition.

Candidate Compounds and Human Amyloid-β1-42 Treatment

Briefly, Aβ₁₋₄₂ peptide (Bachem, ref: H1368 batch 1010533) wasreconstituted in define culture medium at 20 μM (mother solution) andwas slowly shacked at +37° C. for 3 days in dark for aggregation. Thecontrol medium was prepared in the same conditions. After 3 days, thisaggregated human amyloid peptide was used on HBMEC at 2.5 μM diluted incontrol medium (optimal incubation time). The Aβ₁₋₄₂ peptide was added 2hours after HBMEC seeding on matrigel for 18 hours incubation.

One hour after HBMEC seeding on matrigel, test compounds and VEGF-165were solved in culture medium (+0.1% DMSO) and then pre-incubated withHBMEC for 1 hour before the Aβ₁₋₄₂ application (in a final volume perculture well of 100 μl). One hour after test compounds or VEGFincubation (two hours after cell seeding on matrigel), 100 μl of Aβ₁₋₄₂peptide was added to a final concentration of 2.5 μM diluted in controlmedium in presence of test compounds or VEGF (in a 200 μA totalvolume/well), in order to avoid further drug dilutions.

Organization of Cultures Plates

VEGF-165 known to be a pro-angiogenic isoform of VEGF-A, was used forall experiment in this study as reference compound. VEGF-165 is one ofthe most abundant VEGF isoforms involved in angiogenesis. VEGF was usedas reference test compound at 10 nM. The following conditions wereassessed:

-   -   Negative Control: medium alone+0.1% DMSO    -   Intoxication: amyloid-β₁₋₄₂ (2.5 μM) for 18 h    -   Positive control: VEGF-165 (10 nM) (1 reference        compound/culture) 1 hr before the Aβ₁₋₄₂ (2.5 μM) addition for a        18 h incubation time.    -   Test compounds: Test compound 1 hr before the Aβ₁₋₄₂ (2.5 μM)        addition for a 18 h incubation time.        Capillary Network Quantification

Per well, 2 pictures with 4× lens were taken using InCell Analyzer™ 1000(GE Healthcare) in light transmission. All images were taken in the sameconditions. Analysis of the angiogenesis networks was done usingDeveloper software (GE Healthcare). The total length of capillarynetwork was assessed.

Data Processing

All values are expressed as mean±s.e.mean of the 3 cultures (n=6 percondition). Statistic analyses were done on the different conditionsperforming an ANOVA followed by the Dunnett's test when it was allowed(Statview software version 5.0). The values (as %) inserted on thegraphs show the amyloid toxicity evolution. Indeed, the amyloid toxicitywas taken as the 100% and the test compound effect was calculated as a %of this amyloid toxicity.

Results

The results are shown in FIG. 6 and table 3. They demonstrate that thedrugs, alone, induce a substantial protective effect against thetoxicity caused by Aβ peptide 1-42:

-   -   Aminocaproic acid alone, at a low dosage of e.g., 160 nM,        induces strong protective effect;    -   Levosimendan, at a dose as low as 8 nm, induces a strong        protective effect.        II.2 Protection Against the Toxicity of Aβ₁₋₄₂ on Primary        Cortical Neuron Cells        Test Compound and Human Amyloid-β1-42 Treatment

Primary rat cortical neurons are cultured as described previously.

Briefly, Aβ₁₋₄₂ peptide was reconstituted in define culture medium at 40μM (mother solution) and was slowly shacked at +37° C. for 3 days indark for aggregation. The control medium was prepared in the sameconditions.

After 3 days, the solution was used on primary cortical neurons asfollows:

After 10 days of neuron culture, drug was solved in culture medium(+0.1% DMSO) and then pre-incubated with neurons for 1 hour before theAβ₁₋₄₂ application (in a final volume per culture well of 100 μl). Onehour after drug incubation, 100 μl of Aβ₁₋₄₂ peptide was added to afinal concentration of 10 μM diluted in presence of drug, in order toavoid further drug dilutions. Cortical neurons were intoxicated for 24hours. Three separate cultures were performed per condition, 6 wells percondition.

BDNF (50 ng/ml) and Estradiol-13 (100 and 150 nM) were used as positivecontrol and reference compounds respectively. Three separate cultureswill be performed per condition, 12 wells per condition.

Organization of Cultures Plates

Estradiol-β at 100 and 150 nM were used as reference test compound andBDNF at 50 ng/ml was used as a positive control.

Estradiol-β and BDNF were solved in culture medium and pre-incubated for1 h before the aggregated amyloid-β₁₋₄₂ application.

The following conditions were assessed:

-   -   1 CONTROL PLAQUE: 12 wells/condition        -   Negative Control: medium alone+0.1% DMSO        -   Intoxication: amyloid-β₁₋₄₂ (10 μM) for 24 h        -   Positive control: BDNF (50 ng/ml) 1 hr followed by            amyloid-β₁₋₄₂ (10 μM) for 24 h        -   Reference compound: Estradiol (150 nM) 1 hr followed by            amyloid-β₁₋₄₂ (10 μM) for 24 h.    -   DRUG PLATE: 6 wells/condition        -   Negative Control: medium alone+0.1% DMSO        -   Intoxication: amyloid-β₁₋₄₂ (10 μM) for 24 h        -   Drug 1: Drug 1-1 hr followed by amyloid-β₁₋₄₂ (10 μM) for 24            h        -   Drug 2: Drug 2-1 hr followed by amyloid-β₁₋₄₂ (10 μM) for 24            h            Lactate Dehydrogenase (LDH) Activity Assay

24 hours after intoxication, the supernatant was taken off and analyzedwith Cytotoxicity Detection Kit (LDH, Roche Applied Science, ref:11644793001, batch: 11800300). This colorimetric assay for thequantification of cell toxicity is based on the measurement of lactatedehydrogenase (LDH) activity released from the cytosol of dying cellsinto the supernatant.

Data Processing

All values are expressed as mean±s.e.mean of the 3 cultures (n=6 percondition). Statistic analyses were done on the different conditions(ANOVA followed by the Dunnett's test when it was allowed, Statviewsoftware version 5.0).

Results

The results obtained for individual selected drugs in the toxicityassays on primary cortical neuron cells are presented in table 3 and inFIG. 7.

TABLE 3 Protective effect in Protective effect in Aβ₁₋₄₂ intoxicatedAβ₁₋₄₂ intoxicated DRUG NAME neuronal cells HBMC Aminocaproic + acidBaclofen (+/−) + + Carbamazine + Carbenoxolone + Cinacalcet +Cinnarizine + Eplerenone + Etomidate + Fenoldopam + Leflunomide +Levosimendan + + Moxifloxacin + Phenformin + Sulfisoxazole + +Sulodexide + Tadalafil + Terbinafine + Zonisamide +II.3 Effect of Combined Therapies on the Toxicity of Human Aβ₁₋₄₂Peptide on Human HBMEC Cells

The efficacy of drug combinations of the invention is assessed on humancells. The protocol which is used in these assays is the same asdescribed in sections II.1 above.

Results

The following drug combinations are tested on human brain microvascularendothelial cells:

-   -   baclofen and aminocaproic acid,    -   baclofen and levosimendan,    -   aminocaproic acid and sulfisoxazole,    -   aminocaproic acid and terbinafine,    -   aminocaproic acid and levosimendan,    -   levosimendan and sulfisoxazole,    -   levosimendan and terbinafine,    -   eplerenone and levosimendan,    -   eplerenone and sulfisoxazole,    -   eplerenone and fenoldopam,    -   sulodexide and levosimendan,    -   sulodexide and sulfisoxazole,    -   sulodexide and fenoldopam,    -   eplerenone and sulodexide,    -   sulodexide and sulfisoxazole,    -   torasemide and aminocaproic acid, or    -   torasemide and levosimendan.

All of the tested drug combinations give protective effect againsttoxicity of human Aβ₁₋₄₂ peptide in HBMEC model, as shown in Table 4below and exemplified in FIGS. 8 to 13 and 15 to 17.

TABLE 4 Protective effect in Aβ₁₋₄₂ intoxicated DRUG NAME HBMEC cellsbaclofen and aminocaproic acid + baclofen and levosimendan +aminocaproic acid and sulfisoxazole + aminocaproic acid andterbinafine + aminocaproic acid and levosimendan + levosimendan andsulfisoxazole + levosimendan and terbinafine + eplerenone andlevosimendan + eplerenone and sulfisoxazole + eplerenone andfenoldopam + sulodexide and levosimendan + sulodexide andsulfisoxazole + sulodexide and fenoldopam + eplerenone and sulodexide +sulodexide and sulfisoxazole + torasemide and aminocaproic acid +torasemide and levosimendan +III. Levosimendan and Sulfisoxazole Combination Therapy EffectivelyProtects Neurons Against Toxicity of Human Aβ₁₋₄₂

In this example, combination therapy using Levosimendan andSulfisoxazole was assessed for its ability to prevent or reduce thetoxic effects of human Aβ₁₋₄₂. The combination therapy was tested underexperimental conditions disclosed in Example II.1. Human brainmicrovascular endothelial cell cultures were used, as disclosed inII.1., and incubated simultaneously or sequentially with the drugcombination.

The results are presented FIG. 8. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofSulfisoxazole and Levosimendan (FIG. 8A) whereas, at thoseconcentrations, Levosimendan (FIG. 8B) and Sulfisoxazole (FIG. 8C) alonehave no significant effect on intoxication.

IV. Terbinafine and Sulfisoxazole Combination Therapy EffectivelyProtects Neurons Against Toxicity of Human Aβ₁₋₄₂

In this example, combination therapy using Terbinafine and Sulfisoxazolewas assessed for its ability to prevent or reduce the toxic effects ofhuman Aβ₁₋₄₂. The combination therapy was tested under experimentalconditions disclosed in Example II.1. Human brain microvascularendothelial cell cultures were used, as disclosed in II.1., andincubated simultaneously or sequentially with the drug combination.

The results are presented FIG. 9. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofTerbinafine and Sulfisoxazole (FIG. 9A) whereas, at thoseconcentrations, Sulfisoxazole (FIG. 9B) and Terbinafine (FIG. 9C) alonehave no significant effect on intoxication.

V. Levosimendan and Baclofen Combination Therapy Effectively ProtectsNeurons Against Toxicity of Human Aβ₁₋₄₂

In this example, a combination therapy using Levosimendan and baclofenwas assessed for its ability to prevent or reduce the toxic effects ofhuman Aβ₁₋₄₂. The combination therapy was tested under experimentalconditions disclosed in Example II.1. Human brain microvascularendothelial cell cultures were used, as disclosed in II.1., andincubated simultaneously or sequentially with the drug combination.

The results are presented FIG. 10. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofLevosimendan and Baclofen (FIG. 10A) whereas, at those concentrations,Levosimendan (FIG. 10B) and baclofen (FIG. 10C) alone have nosignificant effect on intoxication.

VI. Aminocaproic Acid and Terbinafine Combination Therapy EffectivelyProtects Neurons Against Toxicity of Human Aβ₁₋₄₂

In this example, a combination therapy using Aminocaproic acid andTerbinafine was assessed for its ability to prevent or reduce the toxiceffects of human Aβ₁₋₄₂. The combination therapy was tested underexperimental conditions disclosed in Example II.1. Human brainmicrovascular endothelial cell cultures were used, as disclosed inII.1., and incubated simultaneously or sequentially with the drugcombination.

The results are presented FIG. 11. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofAminocaproic acid and Terbinafine (FIG. 11A) whereas, at thoseconcentrations, Aminocaproic acid (FIG. 11B) and Terbinafine (FIG. 11C)alone have no significant effect on intoxication.

VII. Aminocaproic Acid and Levosimendan Combination Therapy EffectivelyProtects Neurons Against Toxicity of Human Aβ₁₋₄₂

In this example, a combination therapy using Aminocaproic acid andLevosimendan was assessed for its ability to prevent or reduce the toxiceffects of human Aβ₁₋₄₂. The combination therapy was tested underexperimental conditions disclosed in Example II.1. Human brainmicrovascular endothelial cell cultures were used, as disclosed inII.1., and incubated simultaneously or sequentially with the drugcombination.

The results are presented FIG. 12. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofAminocaproic acid and Levosimendan (FIG. 12A) whereas, at thoseconcentrations, Aminocaproic acid (FIG. 12B) and Levosimendan (FIG. 12C)alone have no significant effect on intoxication.

VIII. Terbinafine and Levosimendan Combination Therapy EffectivelyProtects Neurons Against Toxicity of Human Aβ₁₋₄₂

In this example, a combination therapy using Levosimendan andTerbinafine was assessed for its ability to prevent or reduce the toxiceffects of human Aβ₁₋₄₂. The combination therapy was tested underexperimental conditions disclosed in Example II.1. Human brainmicrovascular endothelial cell cultures were used, as disclosed inII.1., and incubated simultaneously or sequentially with the drugcombination.

The results are presented FIG. 13. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofTerbinafine and Levosimendan (FIG. 13A) whereas, at thoseconcentrations, Terbinafine (FIG. 13B) and Levosimendan (FIG. 13C) alonehave no significant effect on intoxication.

IX. Carbamazine and Acamprosate Combination Therapy Effectively ProtectsNeurons Against Toxicity of Human Aβ₁₋₄₂

In this example, a combination therapy using carbamazine and acamprosatewas assessed for its ability to prevent or reduce the toxic effects ofhuman Aβ₁₋₄₂. The combination therapy was tested under experimentalconditions disclosed in Example II.2. Primary rat cortical neuronscultures were used, as disclosed in II.2., and incubated simultaneouslyor sequentially with the drug combination.

The results are presented FIG. 14. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication. This intoxication is significantly prevented by thecombination of carbamazine and acamprosate (FIG. 14).

X. Sulfisoxazole and Sulodexide Combination Therapy Effectively ProtectsEndothelial Cells Against Toxicity of HUMAN Aβ₁₋₄₂

In this example, a combination therapy using sulfisoxazole andsulodexide was assessed for its ability to prevent or reduce the toxiceffects of human Aβ₁₋₄₂. The combination therapy was tested underexperimental conditions disclosed in Example II.1. Human brainmicrovascular endothelial cell cultures were used, as disclosed inII.1., and incubated simultaneously or sequentially with the drugcombination.

The results are presented FIG. 15. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination of sulodexideand sulfisoxazole (FIG. 15A) whereas, at those concentrations,Sulfisoxazole (FIG. 15B) and Sulodexide (FIG. 15C) alone have nosignificant effect on intoxication.

XI. Aminocaproic Acid and Torasemide Combination Therapy EffectivelyProtects Endothelial Cells Against Toxicity of Human Aβ₁₋₄₂

In this example, a combination therapy using Aminocaproic acid andTorasemide was assessed for its ability to prevent or reduce the toxiceffects of human Aβ₁₋₄₂. The combination therapy was tested underexperimental conditions disclosed in Example II.1. Human brainmicrovascular endothelial cell cultures were used, as disclosed inII.1., and incubated simultaneously or sequentially with the drugcombination.

The results are presented FIG. 16. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. With aprevention of 48%, this intoxication is significantly prevented by thecombination of Aminocaproic acid and Torasemide (FIG. 16).

XII. Torasemide and Levosimendan Combination Therapy EffectivelyProtects Endothelial Cells Against Toxicity of Human Aβ₁₋₄₂

In this example, a combination therapy using torasemide and levosimendanwas assessed for its ability to prevent or reduce the toxic effects ofhuman Aβ₁₋₄₂. The combination therapy was tested under experimentalconditions disclosed in Example II.1. Human brain microvascularendothelial cell cultures were used, as disclosed in II.1., andincubated simultaneously or sequentially with the drug combination.

The results are presented FIG. 17. They clearly show that the aggregatedhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) produces a significantintoxication, above 40%, compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination of torasemideand levosimendan (FIG. 17).

XIII. In Vivo Activity

Compounds and their combinations active in in vitro tests are tested inin vivo model of Alzheimer's disease. Overexpression of Alzheimer'sdisease-linked mutant human amyloid beta protein precursor (APP)transgenes has been the most reliable means of promoting deposition ofAbeta in the brains of transgenic mice that served as AD disease modelsin numerous studies. As they age, these mutant APP mice develop robustamyloid pathology and other AD-like features, including decreasedsynaptic density, reactive gliosis, and some cognitive deficits. Manymutant APP mouse models show little evidence of overt neuronal loss andneurofibrillary tangle (NFT) pathology. Mice hemizygous for thisBRI-Abeta42 transgene are viable and fertile with a normal lifespan.Transgenic BRI-Abeta42 mRNA is expressed in a pattern characteristic ofthe mouse prion protein promoter; highest transgene expression levelsare detected in the cerebellar granule cells and hippocampus, followedby the cortex, pons, thalamus, and midbrain. In the transgenic fusionprotein, Abeta1-42 is fused to the C terminus of the BRI protein at thefurin-like cleavage site such that cleavage results in efficientAbeta1-42 secretion into the lumen or extracellular space. Therefore,these mice specifically express the Abeta1-42 isoform. HemizygousBRI-Abeta42 mice accumulate detergent-insoluble amyloid-beta with ageand develop cored plaques in the cerebellum at as early as 3 months ofage. Development of forebrain pathology occurs later, extracellularAbeta plaques are not present consistently in the hippocampus andentorhinal/piriform cortices until 12 months of age. Amyloid betadeposits (cored plaques) can be observed as early as 3 months inmolecular layer of cerebella of transgenic mice and becoming morepronounced with age; occasional extracellular plaques are seen in theentorhinal/piriform cortices and hippocampus at 6 months of age, butaren't consistently found until >12 months of age. Oldest mice showwidespread pathology with cored and diffuse plaques in cerebellum,cortex, hippocampus, and olfactory bulb. Extracellular amyloid plaquesshow dense amyloid cores with radiating fibrils; many bundles ofdystrophic neurites are observed at the periphery of these plaques.Reactive gliosis is associated with plaques.

Drug Treatments

The transgenic Tg (Prnp-ITM2B/APP695*42) A12E mc mice (37) has beenobtained from Jackson Laboratory(http://jaxmice.jax.org/strain/007002.html). Mice founder with thehighest Abeta42 plasma levels, line BRI-Abeta42A (12e), have beenmaintained on a mixed B6C3 background. Adult male transgenic mice havefree access to food and water. In accord with an approved theInstitutional Animal Care and Use Committee protocol, mice are weighedand injected i.p. or force fed once daily for 10 to 20 consecutive weekswith either a control solution (placebo) or drugs of the presentinvention or drug combinations of Table 2, prepared at different doses.

Survival Analysis

Survival rates have been analyzed using Kaplan-Meier methods. Holm-Sidakmethods (post hoc) have been used for all pairwise multiple comparisontests. The extraneous deaths are censored. All comparisons have beenmade between littermates to limit any potentially confounding effectsfrom background strain differences.

Behavioural Tests

Behavioural tests were designed and conducted according to the methodspublished by several authors (38-41).

Spatial Learning and Memory in the Morris Water Maze (MWM)

This experiment is performed in a circular pool, 90 cm in diameter, madeof white plastic and filled with milky colored water. An escapeplatform, 8 cm in diameter, made of clear plastic was submerged 0.5 cmunder the water level. Visual clues are provided by differentgeometrical forms printed in A4-sized letters and placed on the foursurrounding walls (distance from the pool was from 50 to 70 cm). Eachmouse has been given four trials daily (5- to 7-minute interval betweentrials, a total of 16 trials) for 4 days. Each trial has been performedfrom one of four different starting points. The movement of the mice ismonitored using Videotrack Software (View Point). The time taken tolocate the escape platform (escape latency; up to 60 seconds) has beendetermined. After locating the platform the mouse has been allowed tosit on it for 15 seconds. Mice who failed to find the platform within 60seconds have been guided to it and allowed to stay on it for 15 seconds.A latency of 60 seconds is entered into the record for such anoccurrence. All four trials per day have been averaged for statisticalanalysis, except for the first trial on day 1. On day 9 (5 days afterthe last training) mice have been subjected to a 60-second probe trialin which the platform is removed and the mice are allowed to search forit. The time that each animal spent in each quadrant has been recorded(quadrant search time). Several groups of male mice have been used at 3,7, 10, and 12 months. The some few mice have showed freezing behaviour(e.g., lying motionless in the water and refusing to swim) that stronglyinterfered with the test; these animals have been excluded from the dataanalysis. All behavioural tests are conducted under a quiet andlight-reduced environment.

Working Memory Test in Radial Arm Water Maze

This cognitive-based sensitive measure of working memory has beenobtained with the help of the apparatus consisting of a 100 cm-diameterwater-filled pool (also used for the Morris water maze and PlatformRecognition tasks) fitted with an aluminum insert to create sixradially-distributed swim arms. Testing consists of five, 1-min trialsper daily session, for 9-12 consecutive days. At the start of eachsession, a clear submerged platform is positioned at the end of one ofthe six swim arms (randomly-selected, changed daily). For each of thefirst four acquisition trials, the animal is placed into one of thenon-platform containing arms (randomized sequence) and allowed to searchfor the platform. During the 60 s trial, each time the animal entersanother non-platform containing arm, it is gently returned to itsstarting location and an error recorded. After the fourth trial, theanimal is allowed to rest for 30 min, followed by a fifth (retention)trial, which originates in the final non-platform containing swim arm.The number of errors (incorrect arm choices) and escape latency (time toreach platform, maximum 60 s) are recorded for each trial.

Spatial Reference Learning and Memory in Circular Platform Test

This cognitive-based task test is performed with the help of theapparatus that consists of a 69 cm-diameter circular platform having 16“escape” holes spaced equidistantly around the circumference. An escaperefuge is installed beneath one of the holes, and a black curtain, onwhich are placed various visual cues, encircles the platform. The animalis placed in the center of the platform at the start of a single, 5 mintrial and aversive stimuli (bright lights, fan wind) are presented. Thetotal number of errors (head-pokes into non-escape holes) and escapelatency (time to reach escape hole) are recorded.

Recognition Ability in Platform Recognition Test

This cognitive-based search task evaluates object identification andrecognition ability. The target object consists of a 9 cm-diametercircular platform fitted with a 10 cm×40 cm black ensign, which ispositioned 0.8 cm above the surface of the water in a 100 cm-diametercircular pool. Testing consists of four 60 s trials per day on each offour consecutive days. On each day, the target object is placed into adifferent quadrant of the pool for each trial, and the animal isreleased at the same location along the circumference of the pool forall four trials. The total latency (maximum 60 s) is recorded for eachtrial.

Modified Irwin Examination

A comprehensive screen, modified from Irwin is used to determine whetherany of the mice exhibited physiological, behavioural, or sensorimotorimpairments related to their genotype. To explore motor skills,coordination, and muscle strength, the mice are placed on a wire thatwas tightened between two 30-cm-high columns and their ability tobalance on the wire is assessed. In addition, their ability to grasp andhang on the wire with all four paws for at least 5 seconds and to climbback on the wire is determined.

Quantification of Vascular Amyloid Deposition

For quantification of cerebral amyloid angiopathy (CAA), 5 μmparaffin-embedded sections at 30 μm intervals through the parietal orcerebellar cortex leptomeninges are immunostained with biotinylated-Ab9antibody (anti-Aβ1-16, 1:500) overnight at 4° C. (n=5-7 mice pergenotype at each age group, n=6 sections per mouse). Positively stainedblood vessels are visually assessed using modified Vonsattel's scoringsystem (42) The CAA severity score is calculated by multiplying thenumber of CAA vessels with the CAA severity grade.

Histology: Immunohistochemistry and Immunofluorescence

Tg and WT mice from 3 to 12 months are anesthetized and transcardiallyperfused sequentially with 0.9% NaCl and 4% paraformaldehyde in 0.1mol/L phosphatebuffered saline (PBS) (pH 7.4) or 10% formalin and 4%paraformaldehyde in 0.1 mol/L PBS (pH 7.4). Brains and spinal cords areremoved and stored in 4% paraformaldehyde. Some samples are embedded inparaffin and cut on a sliding microtome at a thickness of 10 μm.Cryosections (14 μm) are cut on a cryostat and mounted on chromealum-coated slides. Endogenous peroxidase is quenched by treating thesection with methanol containing 0.3% H2O2 for 30 minutes. Sections areblocked in 10% horse serum. Primary antibodies are used and incubatedovernight at 4° C. in the presence of 1% horse serum. All secondarybiotinylated or fluorescein-, Texas Red-, and AMCA-coupled antibodies,fluorochromes, ABC-kit, and 3,3′-diaminobenzidine as chromogen forperoxidase activity are from Vector Laboratories. Incubation with thesecondary antibody is held at room temperature for 1 hour. All washingsteps (3-10 minutes) and antibody dilution are performed usingphosphate-buffered saline (0.1 mol/L PBS, pH 7.4) or Tris-bufferedsaline (0.01 mol/L Tris, 0.15 mol/L NaCl, pH 7.4). Incubation with theABC complex and detection with 3,3′-diaminobenzidine is carried outaccording to the manufacturer's manual. Hematoxylin counterstaining isperformed according to standard procedures. A minimum of three mice pergenotype, age, and sex is used for each determination (43).

Preparation of Brain Extracts

Brains are rapidly harvested over ice between 90 and 120 min after thefinal injection and frozen to −80° C. The right cerebral hemisphere fromeach mouse is weighed after freezing. Analysis of hemisphere mass bymedian absolute deviation allows us to exclude samples that are beyond 4median absolute deviations from the rest of the set. Cerebralhemispheres are homogenized, and cell lysates containing whole proteinare prepared according to the manufacturer's instructions for enzymaticassay kits (R&D Systems, Inc.). In brief, the brain cortices arehomogenized in 800 μA of low salt containing 1× extraction buffer (R&Dkit) and incubated on ice for 10 min. The homogenates are thencentrifuged at 13,000 g for 15 min at 4° C. The protein concentration ineach sample is estimated according to biuret-derived assay (Pierce).Levels of APP, β40, and β42 are measured by Western immunoblotting andsandwich ELISA techniques. In addition, activities of α, β-, andγ-secretases may be measured from the same extracts.

Assay of Levels of Total APP in Mouse Cerebral Cortex Extracts

An equal-protein amount of brain extracts is loaded in each gel, 30 μgper lane per sample. Each gel contained eight treatments: control; drug17.5 mg/kg dose; and drug 2 in several doses. To minimize intra-gelvariation, each gel contained three sets of all treatment groups. Eachblot is probed with 22C11 antibody. Each blot is also probed with theβ-actin antibody for normalization to transfer efficiency. The intensityof APP band signal is normalized with that of β-actin. Two sample“controls” are loaded in each gel/blot to test for blot to blotvariation. Analysis of blots is performed in two ways: blot wise (n=3),to test for gel to gel variation; and combined blots (n=9 or 10) asdescribed (38-39). Blot-wise analysis with n=3 shows the same trend asthe final analysis with n=9 or 10 does. Results of the combined analysisare presented.

Aβ Sandwich ELISA

For brain Aβ ELISAs, forebrain and hindbrain Aβ levels are determinedindependently, and the olfactory bulb is excluded from analysis. Forplasma Aβ analysis, blood is collected in EDTA-coated tubes aftercardiac puncture. Blood samples are centrifuged at 3000 rpm for 10 minat 4° C., and the plasma is aliquoted and stored at −80° C. until used.Aβ levels are determined by end-specific sandwich ELISAs using Ab9(anti-Aβ1-16 Ab) as the capture Ab for Aβ40, 13.1.1-HRP (anti-Aβ35-40Ab) as the detection Ab for Aβ40, 2.1.3 (anti-Aβ35-42 Ab) as the captureAb for Aβ42, and Ab9-HRP as the detection Ab for Aβ42 (n=5-7 mice pergenotype at each age group). Aβ levels are normalized to the previousresults using the same sets of mice as internal controls to minimizepotential ELISA variability, as described (46).

Western Blotting

Snap-frozen forebrain samples are homogenized inradioimmunoprecipitation assay (RIPA) buffer (Boston BioProducts,Worcester, Mass.) with 1% protease inhibitor mixture (Roche). Thehomogenate is centrifuged at 100,000×g for 1 h at 4° C. Proteinconcentration in supernatants is determined using the BCA protein assay(Pierce). Protein samples (20 μg) are run on Bis-Tris 12% XT gels orBis-Tris 4-12% XT gels (Bio-Rad, Hercules, Calif.) and transferred to0.2 μm nitrocellose membranes. Blots are microwaved for 2 min in 0.1 MPBS twice and probed with Ab 82E1 (anti-Aβ1-16, 1:1000; IBL, Gunma,Japan) and anti-APP C-terminal 20 amino acids (1:1000) as described(46). Blots are stripped and reprobed with anti β-actin (1:1000; Sigma)as a loading control. Relative band intensity is measured using ImageJsoftware.

Quantification of Parenchymal Amyloid Deposition

Hemibrains are immersion fixed in 10% formalin and processed forparaffin embedding. Brain tissue sections (5 μm) were immunostained withanti-total Aβ antibody (Ab). Sections are counterstained withhematoxylin. Six sections per brain through the hippocampus, piriformcortex (bregma, −1.70 to −2.80 mm), or cerebellum (paraflocculus, crusansiform, and simple lobules; bregma, −5.40 to −6.36 mm) are used forquantification (n=5-7 mice per genotype at each age group). The Aβplaque burden is determined using MetaMorph software (Molecular Devices,Palo Alto, Calif.). For quantification of cored plaques, serial sectionsof those analyzed for Aβ burden are stained with thioflavine S (ThioS),and the number of ThioS-positive plaques in the hippocampus,entorhinal/piriform cortex, or the cerebellum is counted. All of theabove analyses are performed in a blinded manner.

Statistical Analysis of In Vivo Data

Results from all experiments are analyzed with STATISTICA 8.0(Statsoft). Aβ levels, amyloid plaque burden, and CAA severity areanalyzed by using ANOVA with the post hoc Holm-Sidak multiple comparisontest or two-tailed Student's t test. If the data set does not meet theparametric test assumptions, either the Kruskal-Wallis test followed bythe post hoc Dunn's multiple comparison or the Mann-Whitney rank sumtest is performed. To test whether the Aβ levels in the bitransgenicmice were consistent with an additive sum of Aβ levels in the singletransgenic littermates, a multiple linear regression with no intercepttest is used. All comparisons are made between littermates. Drugresponse modelling is done excluding the control (0 mg/kg) samples. ED50corresponds to the dose (mg/kg) required to induce a 50% of maximaldrug-induced response in the experiments. It is calculated using theHill equation model for the log of ED50.

In vivo experiments are performed for candidate drug combinations.Positive results on learning and spatial memory are listed in table 5below.

TABLE 5 Results in Drug Morris Water Maze experiment Terbinafine andLevosimendan + Terbinafine and Sulfisoxazole + Baclofen andLevosimendan + Sulfisoxazole and Levosimendan + Aminocaproic acid andLevosimendan + Aminocaproic acid and Terbinafine +

REFERENCES

-   1. Crook R., Verkkoniemi A., et al. (1998). A variant of Alzheimer's    disease with spastic paraparesis and unusual plaques due to deletion    of exon 9 of presenilin 1. Nat. Med. 4(4): 452-5.-   2. Houlden H., Baker M., et al. (2000). Variant Alzheimer's disease    with spastic paraparesis and cotton wool plaques is caused by PS-1    mutations that lead to exceptionally high amyloid-beta    concentrations. Ann Neurol. 48(5): 806-8.-   3. Kwok J. B., Taddei K., et al. (1997). Two novel (M233T and R278T)    presenilin-1 mutations in early-onset Alzheimer's disease pedigrees    and preliminary evidence for association of presenilin-1 mutations    with a novel phenotype. Neuroreport. 8(6): 1537-42.-   4. Verkkoniemi A., Kalimo H., et al. (2001). Variant Alzheimer    disease with spastic paraparesis: neuropathological phenotype. J    Neuropathol Exp Neurol. 60(5): 483-92.-   5. Citron M. (2004). Strategies for disease modification in    Alzheimer's disease. Nat Rev Neurosci. 5(9): 677-85.-   6. Suh Y. H. and Checker F. (2002). Amyloid precursor protein,    presenilins, and alpha-synuclein: molecular pathogenesis and    pharmacological applications in Alzheimer's disease. Pharmacol Rev.    54(3): 469-525.-   7. Blacker D., Albert M. S., et al. (1994). Reliability and validity    of NINCDS-ADRDA criteria for Alzheimer's disease. The National    Institute of Mental Health Genetics Initiative. Arch Neurol. 51(12):    1198-204.-   8. Rossor M. N., Fox N. C., et al. (1996). Clinical features of    sporadic and familial Alzheimer's disease. Neurodegeneration. 5(4):    393-7.-   9. Glenner G. G., Wong C. W., et al. (1984). The amyloid deposits in    Alzheimer's disease: their nature and pathogenesis. Appl Pathol.    2(6): 357-69.-   10. Ballatore C., Lee V. M., et al. (2007). Tau-mediated    neurodegeneration in Alzheimer's disease and related disorders. Nat    Rev Neurosci. 8(9): 663-72.-   11. Bell K. F. and Claudio Cuello A. (2006). Altered synaptic    function in Alzheimer's disease. Eur J. Pharmacol. 545(1): 11-21.-   12. Hardy J. A. and Higgins G. A. (1992). Alzheimer's disease: the    amyloid cascade hypothesis. Science. 256(5054): 184-5.-   13. Braak H. and Braak E. (1991). Neuropathological stageing of    Alzheimer-related changes. Acta Neuropathol. 82(4): 239-59.-   14. Golde T. E. (2005). The Abeta hypothesis: leading us to    rationally-designed therapeutic strategies for the treatment or    prevention of Alzheimer disease. Brain Pathol. 15(1): 84-7.-   15. Hardy J. and Selkoe D. J. (2002). The amyloid hypothesis of    Alzheimer's disease: progress and problems on the road to    therapeutics. Science. 297(5580): 353-6.-   16. Selkoe D. J. (2000). The genetics and molecular pathology of    Alzheimer's disease: roles of amyloid and the presenilins. Neurol    Clin. 18(4): 903-22.-   17. Huang Y. Z., Won S., et al. (2000). Regulation of neuregulin    signaling by PSD-95 interacting with ErbB4 at CNS synapses. Neuron.    26(2): 443-55.-   18. Naruse S., Thinakaran G., et al. (1998). Effects of PS1    deficiency on membrane protein trafficking in neurons. Neuron.    21(5):1213-21.

19. Leeuwen F. N., Kain H. E., et al. (1997). The guanine nucleotideexchange factor Tiam1 affects neuronal morphology; opposing roles forthe small GTPases Rac and Rho. J. Cell Biol. 139(3):797-807.

-   20. Ge G., Fernandez C. A., et al. (2007). Bone morphogenetic    protein 1 processes prolactin to a 17 kDa antiangiogenic factor.    Proc Natl Acad Sci USA. 104(24):10010-5.-   21. Hardie D. G. (2007). AMP-activated/SNF1 protein kinases:    conserved guardians of cellular energy. Nat Rev Mol Cell Biol.    8(10): 774-85.-   22. Reihill J. A., Ewart M. A., et al. (2007). AMP-activated protein    kinase mediates VEGF-stimulated endothelial NO production. Biochem    Biophys Res Commun. 354(4):1084-8.-   23. Ouchi N., Kobayashi H., et al. (2004). Adiponectin stimulates    angiogenesis by promoting cross-talk between AMP-activated protein    kinase and Akt signaling in endothelial cells. J Biol. Chem.    279(2):1304-9.-   24. Hug C., Wang J., et al. (2004). T-cadherin is a receptor for    hexameric and high-molecular-weight forms of Acrp30/adiponectin.    Proc Natl Acad Sci USA. 101(28):10308-13.-   25. English D., Kovala A. T., et al. (1999). Induction of    endothelial cell chemotaxis by sphingosine 1-phosphate and    stabilization of endothelial monolayer barrier function by    lysophosphatidic acid, potential mediators of hematopoietic    angiogenesis. J Hematother Stem Cell Res. 8(6):627-34.-   26. Gorlach A., Klappa P., et al. (2006). The endoplasmic reticulum:    folding, calcium homeostasis, signaling, and redox control. Antioxid    Redox Signal. 8(9-10): 1391-418.-   27. Verkhratsky A. (2004). Endoplasmic reticulum calcium signaling    in nerve cells. Biol Res. 37(4): 693-9.-   28. Cookson M. R. (2003). Neurodegeneration: how does parkin prevent    Parkinson's disease? Curr Biol. 13(13): R522-4.-   29. Sze C. I., Su M., et al. (2004). Down-regulation of WW    domain-containing oxidoreductase induces Tau phosphorylation in    vitro. A potential role in Alzheimer's disease. J Biol. Chem.    279(29): 30498-506.-   30. Walchli S., Curchod M. L., et al. (2000). Identification of    tyrosine phosphatases that dephosphorylate the insulin receptor. A    brute force approach based on “substrate-trapping” mutants. J Biol.    Chem. 275(13):9792-6.-   31. Chang N. S., Doherty J., et al. (2003). JNK1 physically    interacts with WW domain-containing oxidoreductase (WOX1) and    inhibits WOX1-mediated apoptosis. J Biol. Chem. 278(11):9195-202.-   32. D'Orazi G., Cecchinelli B., et al. (2002).    Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser    46 and mediates apoptosis. Nat Cell Biol. 4(1):11-9.-   33. Zhu H., Wu L., et al. (2003). MDM2 and promyelocytic leukemia    antagonize each other through their direct interaction with p53. J    Biol. Chem. 278(49):49286-92.-   34. Rodrigues S., De Wever O., et al. (2007). Opposing roles of    netrin-1 and the dependence receptor DCC in cancer cell invasion,    tumor growth and metastasis. Oncogene. 26(38):5615-25.-   35. Taniguchi Y., Kim S. H., et al. (2003). Presenilin-dependent    “gamma-secretase” processing of deleted in colorectal cancer (DCC).    J Biol. Chem. 278(33):30425-8.-   36. Singer C., Figueroa-Masot X., Batchelor R., and Dorsa D.    Mitogen-activated protein kinase pathway mediates estrogen    neuroprotection after glutamate toxicity in primary cortical    neurons. J. Neuroscience, 1999, 19(7):2455-2463.-   37. McGowan E., et al. (2005) Aβ42 Is Essential for Parenchymal and    Vascular Amyloid Deposition in Mice. Neuron 47: 191-199.-   38. Leighty R. E. et al. (2008) Use of artificial neural networks to    determine cognitive impairment and therapeutic effectiveness in    Alzheimer's transgenic mice. Journal of Neuroscience Methods 167:    358-366-   39. Ashe K H (2001) Learning and memory in transgenic mice modelling    Alzheimer's disease. Learning and Memory 8: 301-308.-   40. Carlson G A, et al. (1997) Genetic modification of the    phenotypes produced by amyloid precursor protein overexpression in    transgenic mice. Human Molecular Genetics 6:1951-1959.-   41. Hsiao K, et al. (1996) Correlative memory deficits, Abeta    elevation, and amyloid plaques in transgenic mice. Science 274:    99-102.-   42. Greenberg S. M. and Vonsattel J. P. (1997) Diagnosis of cerebral    amyloid angiopathy. Sensitivity and specificity of cortical biopsy.    Stroke 28(7):1418-22-   43. Schindowski K. et al. (2006) Alzheimer's Disease-Like Tau    Neuropathology Leads to Memory Deficits and Loss of Functional    Synapses in a Novel Mutated Tau Transgenic Mouse without Any Motor    Deficits. Am J. Pathol. 169: 599-616.-   44. Lahiri D K, et al. (2004) Dietary supplementation with melatonin    reduces levels of amyloid beta-peptides in the murine cerebral    cortex. Journal of Pineal Research 36:224-231.-   45. Basha M R, et al. (2005) The fetal basis of amyloidogenesis:    exposure to lead and latent overexpression of amyloid precursor    protein and beta-amyloid in the aging brain. Journal of Neuroscience    25: 823-829.-   46. Lahiri D. K. et al. (2007) Experimental Alzheimer's Disease Drug    Posiphen[(Phenserine]Lowers Amyloid-betaPeptide Levels in Cell    Culture and Mice. Journal of Pharmacology and experimental    therapeutics 320: 386-396.-   47. Paris D, et al. (2005) Anti-angiogenic activity of the mutant    Dutch A(beta) peptide on human brain microvascular endothelial    cells. Brain Res Mol Brain Res. 136: 212-30.

We claim:
 1. A method of treating a mammalian subject suffering fromAlzheimer's disease (AD) or an AD related disorder, the methodcomprising administering to said subject an effective amount ofaminocaproic acid or levosimendan, salt(s) thereof, or sustained releaseformulation(s) thereof.
 2. The method of claim 1, wherein said methodcomprises administering to said subject an effective amount ofaminocaproic acid or salt(s) thereof, or sustained releaseformulation(s) thereof, and at least one additional compound selectedfrom the group consisting of acamprosate, amlodipine, argatroban,baclofen, cilostazol, cinacalcet, clopidogrel, dyphylline, fenoldopam,leflunomide, mepacrine, methimazole, phenformin, prilocaine, rifabutin,sulfisoxazole, tadalafil, terbinafine, torasemide, cinnarizine,ciclopirox, eplerenone, carbenoxolone, sulodexide, carbamazine,amobarbital, cefotetan, erythrityl tetranitrate, methyclothiazide,risedronate, enprofylline, oxtriphylline, paramethadione, cefmenoxime,aprindine, etomidate, mitiglinide, benidipine, levosimendan, zonisamide,salts thereof and sustained release formulations thereof, for combined,separate or sequential administration.
 3. The method of claim 2, whereinsaid method comprises administering to said subject an effective amountof a composition comprising aminocaproic acid, or salt(s) thereof orsustained release formulation(s) thereof, and at least one additionalcompound selected from the group consisting of baclofen, sulfisoxazole,terbinafine, torasemide, levosimendan, salts thereof and sustainedrelease formulations thereof, for combined, separate or sequentialadministration.
 4. The method of claim 1, wherein said method comprisesadministering to said subject an effective amount of levosimendan, orsalt(s) thereof or sustained release formulation(s) thereof and at leastone additional compound selected from the group consisting ofaminocaproic acid, acamprosate, amlodipine, argatroban, baclofen,cilostazol, cinacalcet, clopidogrel, dyphylline, fenoldopam,leflunomide, mepacrine, methimazole, phenformin, prilocaine, rifabutin,sulfisoxazole, tadalafil, terbinafine, torasemide, cinnarizine,eplerenone, carbenoxolone, sulodexide, carbamazine, ciclopirox,amobarbital, cefotetan, erythrityl tetranitrate, methyclothiazide,risedronate, enprofylline, oxtriphylline, paramethadione, cefmenoxime,aprindine, etomidate, mitiglinide, benidipine, zonisamide, salts thereofand sustained release formulations thereof, for combined, separate orsequential administration.
 5. The method of claim 4, wherein said methodcomprises administering to said subject an effective amount of acomposition comprising levosimendan, or salt(s) thereof or sustainedrelease formulation(s) thereof, and at least one additional compoundselected from the group consisting of aminocaproic acid, baclofen,sulfisoxazole, terbinafine, torasemide, salts thereof and sustainedrelease formulations thereof, for combined, separate or sequentialadministration.
 6. The method of claim 1, wherein said method comprisingadministering: baclofen and aminocaproic acid, baclofen andlevosimendan, aminocaproic acid and sulfisoxazole, aminocaproic acid andterbinafine, aminocaproic acid and levosimendan, levosimendan andsulfisoxazole, levosimendan and terbinafine, torasemide and aminocaproicacid, torasemide and levosimendan, levosimendan and erythrityltetranitrate, levosimendan and zonisamide, or salt(s) thereof orsustained release formulation(s) thereof.
 7. The method of claim 6,wherein said method comprises administering: baclofen and aminocaproicacid, baclofen and levosimendan, aminocaproic acid and sulfisoxazole,aminocaproic acid and terbinafine, aminocaproic acid and levosimendan,torasemide and aminocaproic acid, torasemide and levosimendan,levosimendan and sulfisoxazole, levosimendan and terbinafine, or salt(s)thereof or sustained release formulation(s) thereof.
 8. The method ofclaim 1, wherein aminocaproic acid or levosimendan, salt(s) thereof, orsustained release formulation(s) thereof are administered with apharmaceutically acceptable carrier or excipient.
 9. The method of claim1, wherein aminocaproic acid or levosimendan, salt(s) thereof, orsustained release formulation(s) thereof are administered repeatedly tothe subject.
 10. The method of claim 6, wherein the drug combination isadministered as a combined composition.
 11. The method of claim 6,wherein each drug of the drug combination is administered sequentially.12. The method of claim 6, wherein each drug of the drug combination isadministered separately.
 13. The method of claim 7, wherein the drugcombination is administered as a combined composition.
 14. The method ofclaim 7, wherein each drug of the drug combination is administeredsequentially.
 15. The method of claim 7, wherein each drug of the drugcombination is administered separately.